Provided herein are methods and compositions for depleting targeted nucleic acid sequences from a sample, enriching for sequences of interest from a sample, and/or partitioning of sequences from a sample. The methods and compositions are applicable to biological, clinical, forensic, and environmental samples.
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1. A composition comprising a mixture of DNA targeted sequences for depletion and DNA sequences of interest, wherein the DNA targeted sequences for depletion and the DNA sequences of interest are adapter-ligated, wherein the adapters are ligated to both the 5′ and 3′ ends of both the target sequences for depletion and the sequences of interest, and wherein the DNA targeted sequences for depletion are complexed to a gRNA-CRISPR/Cas system protein complex and wherein the DNA sequences of interest are in the range of about 0.1% to about 50% of the DNA in the composition, wherein the CRISPR/Cas system protein is Cas9, Cpf1, Cas3, Cas8a-c, Cas10, Csel, Csyl, Csn2, Cas4, Csm2, or CmS.
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This application is a continuation of U.S. patent application Ser. No. 15/573,962 filed Jun. 20, 2017, which is a national stage filing under 35 U.S.C. § 371 of International Application No. PCT/US2015/066949, filed Dec. 19, 2015, which claims the priority benefit of U.S. Provisional Application Ser. No. 62/094,980, filed on Dec. 20, 2014, and of U.S. Provisional Application Ser. No. 62/198,097, filed on Jul. 28, 2015. The contents of these applications are hereby incorporated by reference herein in their entireties.
The content of the ASCII text file of the sequence listing named “155949-00089 Sequence Listing.TXT” which is 18.8 kb in size was created on Jul. 30, 2020, and electronically submitted via EFS-Web herewith the application is incorporated herein by reference in its entirety.
Many human clinical DNA samples, or sample libraries such as cDNA libraries derived from RNA, or extracted DNA samples taken from tissue, fluids, or other host material samples contain highly abundant sequences that have little informative value and increase the cost of sequencing. While methods have been developed to deplete these sequences (e.g., via hybridization capture), these methods are often time-consuming and can be inefficient. Moreover hybridization capture often looks to capture the DNA sequences of interest while discarding the remaining sequences. As a result depletion by hybridization capture is not a viable option when the DNA sequences of interest are not known in advance, e.g. when screening a sample to study all microbial or non-host DNA sequences.
While shotgun sequencing of human samples to study microbial DNA can be done, low levels of microbial DNA in many samples has precluded the shotgun sequencing of many complex and/or interesting samples, due to cost. This is true of, for example, a metagenomic analysis of a sample, where the sample contains more than one species of organism (eukaryotic, prokaryotic, or viral organisms). For example, DNA libraries derived from whole human blood often contain >99% human DNA. Therefore, to detect an infectious agent circulating in human blood from shotgun sequencing, one would need to sequence to very high coverage in order to ensure sufficient coverage. Thus much of the cost associated with sequencing high human DNA samples provides relatively little metagenomic data. As a result many human tissue DNA samples are considered unsuitable for metagenomic sequencing merely because the data yield is low compared to the resources required. Thus there is a need in the art to increase microbial DNA yield in high host DNA samples and specifically to increase the percent of microbial DNA being sequenced when sequencing high host endogenous (HHE) DNA samples.
Recent developments in DNA extraction have provided some sequencing techniques to the point that the field of metagenomics has transitioned from focusing on PCR-amplified 16S ribosomal RNA markers to shotgun sequencing of the whole metagenome. However, shotgun sequencing can yield less than desirable results when sequencing HHE DNA samples due to the low percentage of microbial DNA in the overall sample material. Moreover, shotgun sequencing often fails to provide enough information to make an accurate resolution in metagenomic analysis especially when the selected molecules (e.g., 16S ribosomal RNA) represent only a single lineage. Furthermore, 16S ribosomal RNA lineages cannot often differentiate pathogenic from non-pathogenic strains of closely related bacteria, a key goal of clinical metagenomic analysis.
Instead the use of whole genome DNA and RNA sequences is preferred for metagenetic analysis because it provides information from the entire metagenome. Thus there is a need in the art to provide a DNA and RNA sequencing technique for metagenomic analysis in order to derive improved resolution. For example, whole genome analysis of metagenomes from the fecal material of obese and normal weight patients has revealed highly reproducible differences in microbial community structure. These materials tend to have very high microbial DNA content (>99% microbe and <1% human).
In contrast, sequencing libraries derived from many other tissues including human blood, vagina, nasal mucosal membrane, and lung typically contain >90% human and <10% microbial DNA. While samples with <10% microbial DNA can still, with sufficient sequencing, yield enough information for metagenomic analyses, the required amount of sequencing of specimens with less target DNA is costly and thus untenable for many researchers.
Thus there exists a need in the art to achieve a low-cost, efficient method and compositions for metagenomic analyses. Such methods and compositions are provided herein.
All patents, patent applications, publications, documents, web links, and articles cited herein are incorporated herein by reference in their entireties.
Provided herein are methods and compositions for depleting targeted nucleic acid sequences from a sample, enriching for sequences of interest from a sample, and/or partitioning of sequences from a sample. The methods and compositions are applicable to biological, clinical, forensic, and environmental samples.
In one aspect, provided herein is a method of enriching a sample for sequences of interest, comprising: (a) providing a sample comprising sequences of interest and targeted sequences for depletion, wherein the sequences of interest comprise less than 30% of the sample; (b) contacting the sample with a plurality of CRISPR/Cas system protein-gRNA complexes, wherein the gRNAs are complementary to the targeted sequences, and whereby the targeted sequences are cleaved. In any of the embodiments disclosed herein, the method further comprises extracting the sequences of interest and the targeted sequences for depletion from the sample. In any of the embodiments disclosed herein, the method further comprises fragmenting the extracted sequences. In any of the embodiments disclosed herein, the method further comprises adapter ligating the 5′ and 3′ ends of the fragmented extracted sequences. In any of the embodiments disclosed herein, the cleaved targeted sequences are removed by size-exclusion. In any of the embodiments disclosed herein, the cleaved targeted sequences are removed with the use of biotin. In any of the embodiments disclosed herein, the method further comprises amplifying the sequences of interest. In any of the embodiments disclosed herein, the targeted sequences for depletion are followed by a Protospacer Adjacent Motif or (PAM) sequence. In any of the embodiments disclosed herein, the method comprises contacting the sample with at least 102 unique CRISPR/Cas system protein-gRNA complexes. In any of the embodiments disclosed herein, the sample is any one of a biological sample, a clinical sample, a forensic sample or an environmental sample. In any of the embodiments disclosed herein, the sample comprises host nucleic acid sequences targeted for depletion and non-host nucleic acid sequences of interest. In any of the embodiments disclosed herein, the non-host nucleic acid sequences comprise microbial nucleic acid sequences. In any of the embodiments disclosed herein, the microbial nucleic acid sequences are bacterial, viral or eukaryotic parasitic nucleic acid sequences. In any of the embodiments disclosed herein, the sample is contacted with CRISPR/Cas system protein-gRNA complexes, wherein the gRNAs are complementary to DNA corresponding to ribosomal RNA sequences. In any of the embodiments disclosed herein, the sample is contacted with CRISPR/Cas system protein-gRNA complexes, wherein the gRNAs are complementary to mitochondrial DNA. In any of the embodiments disclosed herein, the sample is contacted with CRISPR/Cas system protein-gRNA complexes, wherein the gRNAs are complementary to sequences encoding globin proteins, sequences encoding a transposon, sequences encoding retroviral sequences, sequences comprising telomere sequences, sequences comprising sub-telomeric repeats, sequences comprising centromeric sequences, sequences comprising intron sequences, sequences comprising Alu repeats, sequences comprising SINE repeats, sequences comprising LINE repeats, sequences comprising dinucleic acid repeats, sequences comprising trinucleic acid repeats, sequences comprising tetranucleic acid repeats, sequences comprising poly-A repeats, sequences comprising poly-T repeats, sequences comprising poly-C repeats, sequences comprising poly-G repeats, sequences comprising AT-rich sequences, or sequences comprising GC-rich sequences. In any of the embodiments disclosed herein, the extracted nucleic acids incudes any one of single stranded DNA, double stranded DNA, single stranded RNA, double stranded RNA, cDNA, synthetic DNA, artificial DNA, and DNA/RNA hybrids. In any of the embodiments disclosed herein, the sequences of interest comprise less than 10% of the extracted nucleic acids. In any of the embodiments disclosed herein, the sequences of interest comprise less than 5% of the extracted nucleic acids. In any of the embodiments disclosed herein, the CRISPR/Cas system protein is Cas9, Cpf1, Cas3, Cas8a-c, Cas10, Cse1, Csy1, Csn2, Cas4, Csm2, or Cm5. In any of the embodiments disclosed herein, the CRISPR/Cas system protein is Cas9. In any of the embodiments disclosed herein, the CRISPR/Cas system protein is catalytically dead, for example the catalytically dead CRISPR/Cas system protein is dCas9. In any of the embodiments disclosed herein, the CRISPR/Cas system protein is a CRISPR/Cas system protein nickase, for example Cas9 nickase. In any of the embodiments disclosed herein, the CRISPR/Cas System protein is thermostable. In any of the embodiments disclosed herein, method further comprises amplifying the product of step (b) using adapter-specific PCR. In any of the embodiments disclosed herein, method further comprises treating the product of step (b) with an enzyme that has exonuclease activity. In any of the embodiments disclosed herein, the enzyme is Exonuclease III or BAL-31. In any of the embodiments disclosed herein, method further comprises including the use of positive control target sequences. In any of the embodiments disclosed herein, method further comprises including the use of negative control gRNAs. In any of the embodiments disclosed herein, the sample is selected from whole blood, plasma, serum, tears, saliva, mucous, cerebrospinal fluid, teeth, bone, fingernails, feces, urine, tissue, and a biopsy.
In another aspect, provided herein is a method of enriching a sample comprising: (a) providing a sample comprising mitochondrial DNA and non-mitochondrial DNA, wherein the mitochondrial DNA and non-mitochondrial DNA are adapter-ligated, and wherein the adapters are ligated to the 5′ and 3′ ends of the mitochondrial DNA and non-mitochondrial DNA; (b) contacting the sample with a plurality of CRISPR/Cas system protein-gRNA complexes, wherein the gRNAs are complementary to the mitochondrial DNA, thereby generating mitochondrial DNA adapter ligated only on one end and non-mitochondrial DNA adapter ligated on both the 5′ and 3′ ends; and (c) enriching the sample for non-mitochondrial DNA. In any one of the embodiments disclosed herein, step (c) comprises amplifying the product of step (b) using adapter-specific PCR. In any of the embodiments disclosed herein, step (c) comprises treating the product of step (b) with an enzyme that has exonuclease activity. In any of the embodiments disclosed herein, the enzyme is Exonuclease III or BAL-31. In any of the embodiments disclosed herein, the method is carried out following a method for analyzing genomic DNA comprising treating DNA isolated from a population of cells with an insertional enzyme to produce a plurality of tagged fragments of non-mitochondrial genomic DNA, whereby also generating a residual amount of tagged mitochondrial DNA. In any of the embodiments disclosed herein, the method further comprises extracting the mitochondrial DNA and non-mitochondrial DNA from the sample. In any of the embodiments disclosed herein, the method further comprises fragmenting the extracted sequences. In any of the embodiments disclosed herein, the mitochondrial DNA is removed by size-exclusion. In any of the embodiments disclosed herein, the mitochondrial DNA is removed with the use of biotin. In any of the embodiments disclosed herein, the enriching the sample comprises amplifying the non-mitochondrial DNA. In any of the embodiments disclosed herein, the method comprises contacting the sample with at least 102 unique CRISPR/Cas system protein-gRNA complexes. In any of the embodiments disclosed herein, the CRISPR/Cas system protein is Cas9, Cpf1, Cas3, Cas8a-c, Cas10, Cse1, Csy1, Csn2, Cas4, Csm2, or Cm5. In any of the embodiments disclosed herein, the CRISPR/Cas system protein is Cas9. In any of the embodiments disclosed herein, the CRISPR/Cas system protein is catalytically dead, for example dCas9. In any of the embodiments disclosed herein, the CRISPR/Cas system protein is a CRISPR/Cas system protein nickase, for example Cas9 nickase. In any of the embodiments disclosed herein, the CRISPR/Cas System protein is thermostable. In any of the embodiments disclosed herein, the enriching comprises amplifying the product of step (b) using adapter-specific PCR. In any of the embodiments disclosed herein, the enriching comprises treating the product of step (b) with an enzyme that has exonuclease activity. In any of the embodiments disclosed herein, the enzyme is Exonuclease III or BAL-31.In any of the embodiments disclosed herein, the method further comprises including the use of positive control target sequences. In any of the embodiments disclosed herein, the method further comprises including the use of negative control gRNAs. In any of the embodiments disclosed herein, the sample is selected from whole blood, plasma, serum, tears, saliva, mucous, cerebrospinal fluid, teeth, bone, fingernails, feces, urine, tissue, and a biopsy.
In another aspect, provided herein is a method of enriching a sample comprising: (a) providing a sample comprising nucleic acids from a first genome and nucleic acids from a second genome, wherein the nucleic acids from the first genome are adapter-ligated on their 5′ and 3′ ends and wherein the nucleic acids from the second genome are adapter-ligated on their 5′ and 3′ ends; (b) contacting the sample with a plurality of CRISPR/Cas system protein-gRNA complexes, wherein the gRNAs are complementary to sites targeted in the nucleic acids from the first genome, thereby generating nucleic acids from the first genome adapter ligated only on one end and nucleic acids from the second genome adapter ligated on both the 5′ and 3′ ends; and (c)enriching the sample for nucleic acids from the second genome. In any of the embodiments disclosed herein, the sample further comprises nucleic acids from additional genomes. In any of the embodiments disclosed herein, the first genome is a host genome, and the second genome is a non-host genome. In any of the embodiments disclosed herein, the method comprises contacting the sample with at least 102 unique CRISPR/Cas system protein-gRNA complexes. In any of the embodiments disclosed herein, the nucleic acids are selected from the group consisting of single stranded DNA, single stranded RNA, double stranded DNA, double stranded RNA, DNA/RNA hybrids, cDNA, synthetic DNA, and artificial DNA. In any of the embodiments disclosed herein, the nucleic acids are double stranded DNA. In any of the embodiments disclosed herein, the nucleic acids are DNA, and wherein the contacting of step (b) generates first genome-DNA adapter ligated on the 5′ end but not the 3′ end and second genome DNA adapter ligated on both the 5′ and 3′ ends. In any of the embodiments disclosed herein, the nucleic acids are DNA. In any of the embodiments disclosed herein, the DNA is genomic DNA. In any of the embodiments disclosed herein, the CRISPR/Cas system protein is Cas9, Cpf1, Cas3, Cas8a-c, Cas10, Cse1, Csy1, Csn2, Cas4, Csm2, or Cm5. In any of the embodiments disclosed herein, the CRISPR/Cas system protein is Cas9. In any of the embodiments disclosed herein, the targeted sites in the first genome nucleic acids are followed by a Protospacer Adjacent Motif or (PAM) sequence that can be bound by Cas9. In any of the embodiments disclosed herein, the CRISPR/Cas system protein is catalytically dead, for example dCas9. In any of the embodiments disclosed herein, the CRISPR/Cas system protein is a CRISPR/Cas system protein nickase, for example Cas9 nickase. In any of the embodiments disclosed herein, the CRISPR/Cas system protein is thermostable. In any of the embodiments disclosed herein, the first genome is from an organism selected from the group consisting of a human, cow, horse, sheep, pig, monkey, dog, cat, gerbil, bird, mouse, and rat. In any of the embodiments disclosed herein, the second genome is from a prokaryotic organism. In any of the embodiments disclosed herein, the second genome is from an eukaryotic organism. In any of the embodiments disclosed herein, the second genome is from a parasite. In any of the embodiments disclosed herein, the second genome is from a virus, bacterial, fungus, or protozoa. In any of the embodiments disclosed herein, the adapter-ligated first genome nucleic acids and adapter-ligated second genome nucleic acids range from 50-1000 bp. In any of the embodiments disclosed herein, the second genome nucleic acids comprise less than 50% of the total nucleic acids in the sample. In any of the embodiments disclosed herein, the second genome nucleic acids comprise less than 5% of the total nucleic acids in the sample. In any of the embodiments disclosed herein, the sample is any one of a biological sample, a clinical sample, a forensic sample or an environmental sample. In any of the embodiments disclosed herein, step (c) comprises amplifying the product of step (b) using adapter-specific PCR. In any of the embodiments disclosed herein, step (c) comprises treating the product of step (b) with an enzyme that has exonuclease activity. In any of the embodiments disclosed herein, the enzyme is Exonuclease III or BAL-31. In any of the embodiments disclosed herein, step (c) comprises removing the first genome nucleic acids by size-exclusion. In any of the embodiments disclosed herein, step (c) comprises removing the first genome nucleic acids with the use of biotin. In any of the embodiments disclosed herein, the method further comprises amplifying the product of step (b) using adapter-specific PCR. In any of the embodiments disclosed herein, the method further comprises treating the product of step (b) with an enzyme that has exonuclease activity. In any of the embodiments disclosed herein, the enzyme is Exonuclease III or BAL-31. In any of the embodiments disclosed herein, the method further comprises including the use of positive control target sequences. In any of the embodiments disclosed herein, the method further comprises including the use of negative control gRNAs. In any of the embodiments disclosed herein, the sample is selected from whole blood, plasma, serum, tears, saliva, mucous, cerebrospinal fluid, teeth, bone, fingernails, feces, urine, tissue, and a biopsy.
In another aspect, provided herein is a method of enriching a sample comprising: (a) providing a sample comprising host nucleic acids and non-host nucleic acids, wherein the host nucleic acids and non-host nucleic acids are adapter-ligated, and wherein the adapters are ligated to the 5′ and 3′ ends of the host nucleic acids and the non-host nucleic acids; (b) contacting the sample with a plurality of CRISPR/Cas system protein-gRNA complexes, wherein the gRNAs are complementary to targeted sites in the host nucleic acids, thereby generating host-nucleic acids adapter ligated only on one end and non-host nucleic acids adapter ligated on both the 5′ and 3′ ends; and (c) enriching the sample for non-host nucleic acids. In any of the embodiments disclosed herein, the method comprises contacting the sample with at least 102 unique CRISPR/Cas system protein-gRNA complexes. In any of the embodiments disclosed herein, the nucleic acids are selected from the group consisting of single stranded DNA, single stranded RNA, double stranded DNA, double stranded RNA, DNA/RNA hybrids, cDNA, synthetic DNA, and artificial DNA. In any of the embodiments disclosed herein, the nucleic acids are double stranded DNA. In any of the embodiments disclosed herein, the nucleic acids are DNA, and wherein the contacting of step (b) generates host-DNA adapter ligated on the 5′ end but not the 3′ end and non-host DNA adapter ligated on both the 5′ and 3′ ends. In any of the embodiments disclosed herein, the nucleic acids are DNA. In any of the embodiments disclosed herein, the DNA is genomic DNA. In any of the embodiments disclosed herein, the CRISPR/Cas system protein is Cas9, Cpf1, Cas3, Cas8a-c, Cas10, Cse1, Csy1, Csn2, Cas4, Csm2, or Cm5. In any of the embodiments disclosed herein, the CRISPR/Cas system protein is Cas9. In any of the embodiments disclosed herein, the targeted sites in the host nucleic acids are followed by a Protospacer Adjacent Motif or (PAM) sequence that can be bound by Cas9. In any of the embodiments disclosed herein, the CRISPR/Cas system protein is catalytically dead, for example dCas9. In any of the embodiments disclosed herein, the CRISPR/Cas system protein is a CRISPR/Cas system protein nickase, for example Cas9 nickase. In any of the embodiments disclosed herein, the CRISPR/Cas system protein is thermostable. In any of the embodiments disclosed herein, the host is selected from the group consisting of a human, cow, horse, sheep, pig, monkey, dog, cat, gerbil, bird, mouse, and rat. In any of the embodiments disclosed herein, the non-host is a prokaryotic organism. In any of the embodiments disclosed herein, the non-host is a eukaryotic organism. In any of the embodiments disclosed herein, the non-host is a parasite. In any of the embodiments disclosed herein, the non-host is selected from the group consisting of a virus, bacterial, fungus, and protozoa. In any of the embodiments disclosed herein, the adapter-ligated host nucleic acids and non-host nucleic acids range from 50-1000 bp. In any of the embodiments disclosed herein, the non-host nucleic acids comprise less than 50% of the total nucleic acids in the sample. In any of the embodiments disclosed herein, the non-host nucleic acids comprise less than 5% of the total nucleic acids in the sample. In any of the embodiments disclosed herein, the sample is any one of a biological sample, a clinical sample, a forensic sample or an environmental sample. In any of the embodiments disclosed herein, step (c) comprises amplifying the product of step (b) using adapter-specific PCR. In any of the embodiments disclosed herein, step (c) comprises treating the product of step (b) with an enzyme that has exonuclease activity. In any of the embodiments disclosed herein, the enzyme is Exonuclease III or BAL-31. In any of the embodiments disclosed herein, step (c) comprises removing the host nucleic acids by size-exclusion. In any of the embodiments disclosed herein, step (c) comprises removing the host nucleic acids with the use of biotin. In any of the embodiments disclosed herein, the method further comprises amplifying the product of step (b) using adapter-specific PCR. In any of the embodiments disclosed herein, the method further comprises treating the product of step (b) with an enzyme that has exonuclease activity. In any of the embodiments disclosed herein, the enzyme is Exonuclease III or BAL-31. In any of the embodiments disclosed herein, the method further comprises including the use of positive control target sequences. In any of the embodiments disclosed herein, the method further comprises including the use of negative control gRNAs. In any of the embodiments disclosed herein, the sample is selected from whole blood, plasma, serum, tears, saliva, mucous, cerebrospinal fluid, teeth, bone, fingernails, feces, urine, tissue, and a biopsy.
In another aspect, provided herein is a method of depleting targeted sequences in a sample comprising:(a) providing a sample comprising sequences of interest and targeted sequences for depletion; (b) contacting the sample with a plurality of CRISPR/Cas system protein-gRNA complexes, wherein the gRNAs are complementary to the targeted sequences, thereby generating cleaved targeted sequences; and (c) contacting the product of step (b) with an enzyme that has exonuclease activity. In any of the embodiments disclosed herein, the enzyme is Exonuclease III or BAL-31. In any of the embodiments disclosed herein, the CRISPR/Cas system protein is Cas9. In any of the embodiments disclosed herein, the targeted sequences for depletion comprise sequences encoding globin proteins, sequences encoding a transposon, sequences encoding retroviral sequences, sequences comprising telomere sequences, sequences comprising sub-telomeric repeats, sequences comprising centromeric sequences, sequences comprising intron sequences, sequences comprising Alu repeats, sequences comprising SINE repeats, sequences comprising LINE repeats, sequences comprising dinucleic acid repeats, sequences comprising trinucleic acid repeats, sequences comprising tetranucleic acid repeats, sequences comprising poly-A repeats, sequences comprising poly-T repeats, sequences comprising poly-C repeats, sequences comprising poly-G repeats, sequences comprising AT-rich sequences, or sequences comprising GC-rich sequences. In any of the embodiments disclosed herein, the method comprises contacting the sample with at least 102 unique CRISPR/Cas system protein-gRNA complexes. In any of the embodiments disclosed herein, the sample is selected from whole blood, plasma, serum, tears, saliva, mucous, cerebrospinal fluid, teeth, bone, fingernails, feces, urine, tissue, and a biopsy.
In another aspect, provided herein is a method of generating cleaved targeted sequences in a sample comprising:(a) providing a sample comprising sequences of interest and targeted sequences for cleavage; (b) contacting the sample with a plurality of CRISPR/Cas system protein-gRNA complexes, wherein the gRNAs are complementary to the targeted sequences, thereby generating cleaved targeted sequences; (c) dissociating the CRISPR/Cas system protein from the cleaved targeted sequences; (d) generating additional cleaved targeted sequences; and (e) recovering the uncut sequences of interest. In any of the embodiments disclosed herein, the CRISPR/Cas system protein is thermostable. In any of the embodiments disclosed herein, the thermostable CRISPR/Cas system protein is thermostable Cas9. In any of the embodiments disclosed herein, the dissociating of the CRISPR/Cas system protein from the cleaved targeted sequences is achieved by elevating the temperature of the mixture of step (b) to at least 75°. In any of the embodiments disclosed herein, the generating of additional cleaved targeted sequences is achieved by lowering the temperature of the mixture of step (b) to at least 50°. In any of the embodiments disclosed herein, step (e) comprises amplifying the product of step (d) using adapter-specific PCR. In any of the embodiments disclosed herein, step (e) comprises treating the product of step (d) with an enzyme that has exonuclease activity. In any of the embodiments disclosed herein, the enzyme is Exonuclease III or BAL-31. In any of the embodiments disclosed herein, the CRISPR/Cas system protein is Cas9. In any of the embodiments disclosed herein, the targeted sequences for cleavage comprise sequences encoding globin proteins, sequences encoding a transposon, sequences encoding retroviral sequences, sequences comprising telomere sequences, sequences comprising sub-telomeric repeats, sequences comprising centromeric sequences, sequences comprising intron sequences, sequences comprising Alu repeats, sequences comprising SINE repeats, sequences comprising LINE repeats, sequences comprising dinucleic acid repeats, sequences comprising trinucleic acid repeats, sequences comprising tetranucleic acid repeats, sequences comprising poly-A repeats, sequences comprising poly-T repeats, sequences comprising poly-C repeats, sequences comprising poly-G repeats, sequences comprising AT-rich sequences, or sequences comprising GC-rich sequences. In any of the embodiments disclosed herein, the method comprises contacting the sample with at least 102 unique CRISPR/Cas system protein-gRNA complexes. In any of the embodiments disclosed herein, the sample is selected from whole blood, plasma, serum, tears, saliva, mucous, cerebrospinal fluid, teeth, bone, fingernails, feces, urine, tissue, and a biopsy.
In another aspect, provided herein is a method of depleting targeted sequences in a sample comprising:(a)providing a sample comprising sequences of interest and targeted sequences for cleavage; (b) contacting the sample with a plurality of CRISPR/Cas system protein-gRNA complexes, wherein the gRNAs are complementary to the targeted sequences, thereby generating cleaved targeted sequences, and wherein the CRISPR/Cas system protein is thermostable; (c) elevating the temperature of the mixture of step (b) to at least 75°; (d) lowering the temperature of the mixture of step (b) at least 50°; (e) repeating steps (c) and (d) at least once; and (f) recovering the uncut sequences of interest. In any of the embodiments disclosed herein, step (f) comprises amplifying the product of step (e) using adapter-specific PCR. In any of the embodiments disclosed herein, step (f) comprises treating the product of step (e) with an enzyme that has exonuclease activity. In any of the embodiments disclosed herein, the enzyme is Exonuclease III or BAL-31. In any of the embodiments disclosed herein, the CRISPR/Cas system protein is Cas9. In any of the embodiments disclosed herein, the targeted sequences for cleavage comprise sequences encoding globin proteins, sequences encoding a transposon, sequences encoding retroviral sequences, sequences comprising telomere sequences, sequences comprising sub-telomeric repeats, sequences comprising centromeric sequences, sequences comprising intron sequences, sequences comprising Alu repeats, sequences comprising SINE repeats, sequences comprising LINE repeats, sequences comprising dinucleic acid repeats, sequences comprising trinucleic acid repeats, sequences comprising tetranucleic acid repeats, sequences comprising poly-A repeats, sequences comprising poly-T repeats, sequences comprising poly-C repeats, sequences comprising poly-G repeats, sequences comprising AT-rich sequences, or sequences comprising GC-rich sequences. In any of the embodiments disclosed herein, the method comprises contacting the sample with at least 102 unique CRISPR/Cas system protein-gRNA complexes. In any of the embodiments disclosed herein, the sample is selected from whole blood, plasma, serum, tears, saliva, mucous, cerebrospinal fluid, teeth, bone, fingernails, feces, urine, tissue, and a biopsy.
In another aspect, provided herein is a method for serially depleting targeted nucleic acids in a sample comprising:(a) providing a sample comprising host nucleic acids and non-host nucleic acids, wherein the non-host nucleic acids comprise nucleic acids from at least one known non-host organism and nucleic acids from at least one unknown non-host organism; (b) contacting the sample with a plurality of CRISPR/Cas system protein-gRNA complexes, wherein the gRNAs are configured to hybridize to targeted sequences in the host nucleic acids, whereby a portion of the host nucleic acids are cleaved; (c) contacting the sample with a plurality of CRISPR/Cas system protein-gRNA complexes, wherein the gRNAs are configured to hybridize to targeted sequences in the at least one known non-host nucleic acids, whereby a portion of the at least one known non-host nucleic acids are cleaved; and (d) isolating the nucleic acids from the unknown non-host organism. In any of the embodiments disclosed herein, the method comprises contacting the sample with at least 102 unique CRISPR/Cas system protein-gRNA complexes configured to hybridize to targeted sequences in the host nucleic acids. In any of the embodiments disclosed herein, the method comprises contacting the sample with at least 102 unique CRISPR/Cas system protein-gRNA complexes configured to hybridize to targeted sequences in the at least one known non-host nucleic acids. In any of the embodiments disclosed herein, the sample is selected from whole blood, plasma, serum, tears, saliva, mucous, cerebrospinal fluid, teeth, bone, fingernails, feces, urine, tissue, and a biopsy. In any of the embodiments disclosed herein, the nucleic acids from an unknown non-host organism comprise less than 5% of the total nucleic acids in the sample. In any of the embodiments disclosed herein, the host is a human. In any of the embodiments disclosed herein, the at least one known non-host organism is a species of Streptococcus.
In another aspect, provided herein is a method for analyzing genomic DNA, comprising:(a) treating DNA isolated from a population of cells from a sample with an insertional enzyme to produce a plurality of tagged fragments of non-mitochondrial genomic DNA, whereby also generating a residual amount of tagged mitochondrial DNA; (b) enriching the product of step (a) for non-mitochondrial DNA according to any of the relevant methods provided herein. For example, such a method of enriching a sample can comprise: (a) providing a sample comprising mitochondrial DNA and non-mitochondrial DNA, wherein the mitochondrial DNA and non-mitochondrial DNA are adapter-ligated, and wherein the adapters are ligated to the 5′ and 3′ ends of the mitochondrial DNA and non-mitochondrial DNA; (b) contacting the sample with a plurality of CRISPR/Cas system protein-gRNA complexes, wherein the gRNAs are complementary to the mitochondrial DNA, thereby generating mitochondrial DNA adapter ligated only on one end and non-mitochondrial DNA adapter ligated on both the 5′ and 3′ ends; and (c) enriching the sample for non-mitochondrial DNA. In any of the embodiments disclosed herein, the method further comprises (a) sequencing at least some of the tagged fragments to produce a plurality of sequence reads; and (b) making an epigenetic map of a region of the genome of said cells by mapping information obtained from the sequence reads to the region. In any of the embodiments disclosed herein, the sample is selected from whole blood, plasma, serum, tears, saliva, mucous, cerebrospinal fluid, teeth, bone, fingernails, feces, urine, tissue, and a biopsy.
In another aspect, provided herein is a composition comprising a mixture of DNA from a first genome and DNA from a second genome, wherein the first genome DNA and the second genome DNA are adapter-ligated, and wherein the first genome DNA is complexed to a gRNA-CRISPR/Cas system protein complex. In any of the embodiments disclosed herein, the first genome is from a host organism and the second genome is from a non-host organism. In any of the embodiments disclosed herein, the CRISPR/Cas system protein is Cas9. In any of the embodiments disclosed herein, the CRISPR/Cas system protein is thermostable.
In another aspect, provided herein are kits. In another aspect, provided herein is a kit comprising: (a) CRISPR/Cas system protein; and (b) gRNAs, wherein the gRNAs are complementary to mitochondrial DNA. In another aspect, provided herein is a kit comprising (a) a CRISPR/Cas system protein; (b) gRNAs, wherein the gRNAs are complementary to a target of interest; and (c) an enzyme having exonuclease activity. In another aspect, provided herein is a kit comprising (a) a CRISPR/Cas system protein, wherein the CRISPR/Cas system protein is thermostable; and (b) gRNAs, wherein the gRNAs are complementary to a target of interest. In another aspect, provided herein is a kit comprising (a) a CRISPR/Cas system protein; and (b) a first set of gRNAs, wherein the gRNAs are complementary to target sequences of interest; and (c) a control set of reagents. In another aspect, provided herein is a kit comprising: (a) reagents for isolating DNA from a population of cells; (b) an insertional enzyme; (c) a CRISPR/Cas system protein; and (d) a plurality of gRNAs, wherein the gRNAs are complementary to mitochondrial DNA. In any of the kits disclosed herein, the kit further comprises a collection of Y-shaped adapters or poly-G adapters. In any of the kits disclosed herein, the CRISPR/Cas system protein is Cas9. In any of the kits disclosed herein, the kits further comprise a positive control set of reagents, for example the positive control set of reagents comprise a collection of nucleic acid fragments, wherein the fragments comprise the target sequences of interest, to which the gRNAs are at least 85% complementary. In any of the kits disclosed herein, the kits further comprise a negative control set of reagents, for example, the negative control set of reagents comprise a second set of gRNAs, wherein the second set of gRNAs exhibit reduced binding to the target sequences of interest, as compared to the first set of gRNAs; or for example the negative control set of reagents can comprise a collection of nucleic acid fragments, wherein the fragments are no more than 90% complementary to the first set of gRNAs. In any one of the kits disclosed herein, the kit further comprises at least 102 unique gRNAs.
In another aspect the invention provides a method of depleting targeted sequences in a sample comprising: providing a sample comprising sequences of interest and targeted sequences for depletion; and contacting the sample with a plurality of CRISPR/Cas system protein-gRNA complexes, wherein the gRNAs are complementary to the targeted sequences, thereby generating cleaved targeted sequences. In a related aspect, the invention provides a method for one of depletion and partitioning of targeted sequences in a sample comprising: providing nucleic acids extracted from a sample, wherein the extracted nucleic acid sequences comprise sequences of interest and targeted sequences for one of depletion and partitioning; providing a plurality of guide RNAs (gRNA)-CRISPR/Cas system protein complexes, wherein the gRNAs are configured to hybridize to different targeted sequences; mixing the nucleic acids with the gRNA-CRISPR/Cas system protein complexes, wherein at least a portion of the plurality of gRNA-CRISPR/Cas system protein complexes hybridizes to the targeted sequences. In any one of the embodiments disclosed herein, the method further comprises extracting the nucleic acid sequences from the sample. In any one of the embodiments disclosed herein the CRISPR/Cas system protein is Cas9. In any one of the embodiments disclosed herein, the method further comprises fragmenting the extracted nucleic acids. In any one of the embodiments disclosed herein, the method further comprises adapter ligating the 5′ and 3′ ends of the fragmented extracted nucleic acids. In any one of the embodiments disclosed herein, the method further comprises incubating the mixture to cleave the targeted sequences. In any one of the embodiments disclosed herein, each sequence targeted for depletion is followed by a Protospacer Adjacent Motif or (PAM) sequence that can be bound by a Cas9 protein derived from a bacterial species but wherein the sequences of interest (for enrichment) are not targeted by the gRNAs. In any one of the embodiments disclosed herein, the Cas9 protein comprises a catalytically dead Cas9 that includes an affinity tag previously attached thereto, further comprising partitioning the mixture into a first portion that includes the complementary target specific nucleic sequence present in the mixture that include a guide RNA/Cas9 complex and a second portion that includes fragmented extracted nucleic acid sequences that are not bound by a guide RNA/Cas9 complex, wherein the partitioning is performed using the affinity tag. In any one of the embodiments disclosed herein, the cleaved extracted nucleic acid sequences are smaller than the fragmented extracted nucleic acid sequences that have not been cleaved, the method further comprising, removing the cleaved extracted nucleic acid sequences from the mixture by size selective exclusion. In any one of the embodiments disclosed herein, the method further comprises amplifying the fragmented extracted nucleic acid sequences that have not been cleaved. In any one of the embodiments disclosed herein, the method further comprises analyzing each of the first and second portions by amplification and sequencing. In any one of the embodiments disclosed herein, the sample is any one of a biological sample, a clinical sample, a forensic sample or an environmental sample. In any one of the embodiments disclosed herein, the extracted nucleic acids comprise host nucleic acid sequences and non-host nucleic acid sequences. In any one of the embodiments disclosed herein, the non-host nucleic acid sequences comprise microbial nucleic acid sequences. In any one of the embodiments disclosed herein, the microbial nucleic acid sequences include any one of bacterial, viral and eukaryotic parasitic nucleic acid sequences. In any one of the embodiments disclosed herein, at least a portion of the host nucleic acid sequences are included in the sequences being targeted for one of depletion and partitioning. In any one of the embodiments disclosed herein, substantially all of the host nucleic acid sequences are included in the sequences being targeted for one of depletion and partitioning. In any one of the embodiments disclosed herein, the extracted nucleic acids comprise a plurality of different ribosomal RNA sequences and the plurality of different target specific gRNAs comprise at least a portion of the target nucleic acid sequences configured to hybridize to a portion of the plurality of different ribosomal RNA sequences. In any one of the embodiments disclosed herein, the extracted nucleic acid sequences comprise a plurality of different mitochondrial nucleic acid sequences and the plurality of different target specific gRNAs comprise one or more target nucleic acid sequence configured to hybridize to a portion of the plurality of different mitochondrial nucleic acid sequences. In any one of the embodiments disclosed herein, the extracted nucleic acid sequences comprise a plurality of different repetitive nucleic acid sequences in the nucleic acid sequences being targeted for depletion and the plurality of different target specific gRNAs comprise at least one target nucleic acid sequence that can hybridize to a portion of the plurality of different repetitive nucleic acid sequences. In any one of the embodiments disclosed herein, the extracted nucleic acids incudes any one of single stranded DNA, double stranded DNA, single stranded RNA, double stranded RNA, cDNA, synthetic DNA, artificial DNA, and DNA/RNA hybrids. In any one of the embodiments disclosed herein, the sequences of interest comprise less than 50% of the extracted nucleic acids. In any one of the embodiments disclosed herein, the sequences of interest comprise less than 5% of the extracted nucleic acids.
In another aspect the invention provides a guide RNA library comprising a plurality of target-specific guide RNAs, each configured to hybridize to a selected target nucleic acid sequence, wherein each guide RNA comprises a sequence that can be bound by a Cas9 protein. In any one of the embodiments disclosed herein, each selected target nucleic acid sequence is immediately followed by a protospacer adjacent motif. In any one of the embodiments disclosed herein, target specific gRNAs include gRNAs suitable for hybridizing with selected target nucleic acid sequences in the human genome. In any one of the embodiments disclosed herein, the human genome includes nucleic acid sequences comprising a plurality of different repetitive nucleic acid sequences and the target-specific gRNAs includes guide elements suitable for hybridizing with each of the plurality of different repetitive nucleic acid sequences.
In another aspect, the technology disclosed herein relates to methods and systems for selective or targeted depletion and/or selective or targeted partitioning of nucleic acid samples using a Cas9-enzyme-mediated method.
In another aspect, the technology disclosed herein relates to forming a library of gRNAs wherein each gRNA is suitable to hybridize to a nucleic acid sequence being targeted for removal from the overall nucleic acid sample wherein the sequence being targeted is followed by a Protospacer Adjacent Motif (PAM) sequence. By mixing the gRNAs with nucleic acid samples and a Cas9 protein derived from a bacterial species the gRNAs hybridize to the nucleic acid sequence being targeted for removal and the Cas9 enzyme forms a guide RNA/Cas9 complex that bonds to the nucleic acid sequence being targeted for removal. Thereafter the guide RNA/Cas9 complex can be incubated to cut the targeted nucleic acid sequences so that cut sequences can be separated from uncut nucleic acid sequences or so that targeted sequences that are bonded by a guide RNA/Cas9 complex can be partitioned from DNA sequences that are not bonded to by a guide RNA/Cas9 complex.
In another aspect, the technology disclosed herein is also useful for depleting host molecules from nucleic acid samples that contain low levels (e.g. <50%) of non-host nucleic acids.
In another aspect, the technology disclosed herein comprises: 1) combining a genomic DNA sample or sequencing library with a mixture of Cas9-gRNA complexes, wherein the Cas9-gRNA complexes comprise a Cas9 protein and Cas9-associated gRNAs that are complementary to a pre-defined site in the genome; and b) incubating the reaction mixture to cut only the target regions. In the embodiment in which a sequencing library is targeted, Cas9 cutting would separate the two sequencing adapters and hence make these fragments unable to be amplified. In the embodiment in which genomic DNA is targeted, Cas9 cutting would make these fragments small enough to be removed by size selection.
Definitions
Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described.
The headings provided herein are not limitations of the various aspects or embodiments of the invention. Accordingly, the terms defined immediately below are more fully defined by reference to the specification as a whole.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Singleton, et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 2D ED., John Wiley and Sons, New York (1994), and Hale & Markham, THE HARPER COLLINS DICTIONARY OF BIOLOGY, Harper Perennial, N.Y. (1991) provide one of skill with the general meaning of many of the terms used herein. Still, certain terms are defined below for the sake of clarity and ease of reference.
Numeric ranges are inclusive of the numbers defining the range.
The term “sample” as used herein relates to a material or mixture of materials, typically, although not necessarily, in liquid form, containing one or more analytes of interest.
The term “nucleic acid sample,” as used herein denotes a sample containing nucleic acids. Nucleic acid samples used herein may be complex in that they contain multiple different molecules that contain sequences. Genomic DNA from a mammal (e.g., mouse or human) are types of complex samples. Complex samples may have more then 104, 105, 106 or 107 different nucleic acid molecules. A DNA target may originate from any source such as genomic DNA, cDNA, or an artificial DNA construct. Any sample containing nucleic acid, e.g., genomic DNA made from tissue culture cells or a sample of tissue may be employed herein.
The term “nucleotide” is intended to include those moieties that contain not only the known purine and pyrimidine bases, but also other heterocyclic bases that have been modified. Such modifications include methylated purines or pyrimidines, acylated purines or pyrimidines, alkylated riboses or other heterocycles. In addition, the term “nucleotide” includes those moieties that contain hapten or fluorescent labels and may contain not only conventional ribose and deoxyribose sugars, but other sugars as well. Modified nucleosides or nucleotides also include modifications on the sugar moiety, e.g., wherein one or more of the hydroxyl groups are replaced with halogen atoms or aliphatic groups, or are functionalized as ethers, amines, or the like.
The term “nucleic acids” and “polynucleotides” are used interchangeably herein. Polynucleotide is used to describe a nucleic acid polymer of any length, e.g., greater than about 2 bases, greater than about 10 bases, greater than about 100 bases, greater than about 500 bases, greater than 1000 bases, up to about 10,000 or more bases composed of nucleotides, e.g., deoxyribonucleotides or ribonucleotides, and may be produced enzymatically or synthetically (e.g., PNA as described in U.S. Pat. No. 5,948,902 and the references cited therein) which can hybridize with naturally occurring nucleic acids in a sequence specific manner analogous to that of two naturally occurring nucleic acids, e.g., can participate in Watson-Crick base pairing interactions. Naturally-occurring nucleotides include guanine, cytosine, adenine and thymine (G, C, A and T, respectively). DNA and RNA have a deoxyribose and ribose sugar backbone, respectively, whereas PNA's backbone is composed of repeating N-(2-aminoethyl)-glycine units linked by peptide bonds. In PNA various purine and pyrimidine bases are linked to the backbone by methylene carbonyl bonds. A locked nucleic acid (LNA), often referred to as inaccessible RNA, is a modified RNA nucleotide. The ribose moiety of an LNA nucleotide is modified with an extra bridge connecting the 2′ oxygen and 4′ carbon. The bridge “locks” the ribose in the 3′-endo (North) conformation, which is often found in the A-form duplexes. LNA nucleotides can be mixed with DNA or RNA residues in the oligonucleotide whenever desired. The term “unstructured nucleic acid,” or “UNA,” is a nucleic acid containing non-natural nucleotides that bind to each other with reduced stability. For example, an unstructured nucleic acid may contain a G′ residue and a C′ residue, where these residues correspond to non-naturally occurring forms, i.e., analogs, of G and C that base pair with each other with reduced stability, but retain an ability to base pair with naturally occurring C and G residues, respectively. Unstructured nucleic acid is described in US20050233340, which is incorporated by reference herein for disclosure of UNA.
The term “oligonucleotide” as used herein denotes a single-stranded multimer of nucleotides.
Unless otherwise indicated, nucleic acids are written left to right in 5′ to 3′ orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively.
The term “cleaving,” as used herein, refers to a reaction that breaks the phosphodiester bonds between two adjacent nucleotides in both strands of a double-stranded DNA molecule, thereby resulting in a double-stranded break in the DNA molecule.
The term “cleavage site, as used herein, refers to the site at which a double-stranded DNA molecule has been cleaved.
The term “hybridization” refers to the process by which a strand of nucleic acid joins with a complementary strand through base pairing as known in the art. A nucleic acid is considered to be “selectively hybridizable” to a reference nucleic acid sequence if the two sequences specifically hybridize to one another under moderate to high stringency hybridization and wash conditions. Moderate and high stringency hybridization conditions are known (see, e.g., Ausubel, et al., Short Protocols in Molecular Biology, 3rd ed., Wiley & Sons 1995 and Sambrook et al., Molecular Cloning: A Laboratory Manual, Third Edition, 2001 Cold Spring Harbor, N.Y.). One example of high stringency conditions includes hybridization at about 42° C. in 50% formamide, 5×SSC, 5×Denhardt's solution, 0.5% SDS and 100 μg/ml denatured carrier DNA followed by washing two times in 2×SSC and 0.5% SDS at room temperature and two additional times in 0.1×SSC and 0.5% SDS at 42° C.
The term “duplex,” or “duplexed,” as used herein, describes two complementary polynucleotides that are base-paired, i.e., hybridized together.
The term “amplifying” as used herein refers to generating one or more copies of a target nucleic acid, using the target nucleic acid as a template.
The term “depleting,” with respect to a genome, refers to the removal of one part of the genome from the remainder of the genome to produce a product that is isolated from the remainder of the genome. The term “depleting” also encompasses removal of DNA from one species while retaining DNA from another species.
The term “genomic region,” as used herein, refers to a region of a genome, e.g., an animal or plant genome such as the genome of a human, monkey, rat, fish or insect or plant. In certain cases, an oligonucleotide used in the method described herein may be designed using a reference genomic region, i.e., a genomic region of known nucleotide sequence, e.g., a chromosomal region whose sequence is deposited at NCBI's Genbank database or other databases, for example.
The term “genomic sequence,” as used herein, refers to a sequence that occurs in a genome. Because RNAs are transcribed from a genome, this term encompasses sequence that exist in the nuclear genome of an organism, as well as sequences that are present in a cDNA copy of an RNA (e.g., an mRNA) transcribed from such a genome.
The term “genomic fragment,” as used herein, refers to a region of a genome, e.g., an animal or plant genome such as the genome of a human, monkey, rat, fish or insect or plant. A genomic fragment may be an entire chromosome, or a fragment of a chromosome. A genomic fragment may be adapter ligated (in which case it has an adapter ligated to one or both ends of the fragment, or to at least the 5′ end of a molecule), or may not be adapter ligated.
In certain cases, an oligonucleotide used in the method described herein may be designed using a reference genomic region, i.e., a genomic region of known nucleotide sequence, e.g., a chromosomal region whose sequence is deposited at NCBI's Genbank database or other databases, for example. Such an oligonucleotide may be employed in an assay that uses a sample containing a test genome, where the test genome contains a binding site for the oligonucleotide.
The term “ligating,” as used herein, refers to the enzymatically catalyzed joining of the terminal nucleotide at the 5′ end of a first DNA molecule to the terminal nucleotide at the 3′ end of a second DNA molecule.
If two nucleic acids are “complementary,” each base of one of the nucleic acids base pairs with corresponding nucleotides in the other nucleic acid. The term “complementary” and “perfectly complementary” are used synonymously herein.
The term “separating,” as used herein, refers to physical separation of two elements (e.g., by size or affinity, etc.) as well as degradation of one element, leaving the other intact.
In a cell, DNA usually exists in a double-stranded form, and as such, has two complementary strands of nucleic acid referred to herein as the “top” and “bottom” strands. In certain cases, complementary strands of a chromosomal region may be referred to as “plus” and “minus” strands, the “first” and “second” strands, the “coding” and “noncoding” strands, the “Watson” and “Crick” strands or the “sense” and “antisense” strands. The assignment of a strand as being a top or bottom strand is arbitrary and does not imply any particular orientation, function or structure. Until they become covalently linked, the first and second strands are distinct molecules. For ease of description, the “top” and “bottom” strands of a double-stranded nucleic acid in which the top and bottom strands have been covalently linked will still be described as the “top” and “bottom” strands. In other words, for the purposes of this disclosure, the top and bottom strands of a double-stranded DNA do not need to be separated molecules. The nucleotide sequences of the first strand of several exemplary mammalian chromosomal regions (e.g., BACs, assemblies, chromosomes, etc.) is known, and may be found in NCBI's Genbank database, for example.
The term “top strand,” as used herein, refers to either strand of a nucleic acid but not both strands of a nucleic acid. When an oligonucleotide or a primer binds or anneals “only to a top strand,” it binds to only one strand but not the other. The term “bottom strand,” as used herein, refers to the strand that is complementary to the “top strand.” When an oligonucleotide binds or anneals “only to one strand,” it binds to only one strand, e.g., the first or second strand, but not the other strand. If an oligonucleotide binds or anneals to both strands of a double-stranded DNA, the oligonucleotide may have two regions, a first region that hybridizes with the top strand of the double-stranded DNA, and a second region that hybridizes with the bottom strand of the double-stranded DNA.
The term “double-stranded DNA molecule” refers to both double-stranded DNA molecules in which the top and bottom strands are not covalently linked, as well as double-stranded DNA molecules in which the top and bottom stands are covalently linked. The top and bottom strands of a double-stranded DNA are base paired with one other by Watson-Crick interactions.
The term “denaturing,” as used herein, refers to the separation of at least a portion of the base pairs of a nucleic acid duplex by placing the duplex in suitable denaturing conditions. Denaturing conditions are well known in the art. In one embodiment, in order to denature a nucleic acid duplex, the duplex may be exposed to a temperature that is above the Tm of the duplex, thereby releasing one strand of the duplex from the other. In certain embodiments, a nucleic acid may be denatured by exposing it to a temperature of at least 90° C. for a suitable amount of time (e.g., at least 30 seconds, up to 30 mins). In certain embodiments, fully denaturing conditions may be used to completely separate the base pairs of the duplex. In other embodiments, partially denaturing conditions (e.g., with a lower temperature than fully denaturing conditions) may be used to separate the base pairs of certain parts of the duplex (e.g., regions enriched for A-T base pairs may separate while regions enriched for G-C base pairs may remain paired). Nucleic acid may also be denatured chemically (e.g., using urea or NaOH).
The term “genotyping,” as used herein, refers to any type of analysis of a nucleic acid sequence, and includes sequencing, polymorphism (SNP) analysis, and analysis to identify rearrangements.
The term “sequencing,” as used herein, refers to a method by which the identity of consecutive nucleotides of a polynucleotide are obtained.
The term “next-generation sequencing” refers to the so-called parallelized sequencing-by-synthesis or sequencing-by-ligation platforms, for example, those currently employed by Illumina, Life Technologies, and Roche, etc. Next-generation sequencing methods may also include nanopore sequencing methods or electronic-detection based methods such as Ion Torrent technology commercialized by Life Technologies.
The term “host nucleic acids” refers to nucleic acids that originate from the multicellular eukaryotic subject from which the sample was obtained. Host nucleic acids can be, e.g., plant or animal, including mammals particularly humans. The term “host nucleic acids” includes nuclear nucleic acids as well as nucleic acids present in other organelles, e.g., the mitochondria and chloroplast (if the host is a plant), but not the nucleic acids from microbes that often grow on or in the subject.
The term “non-host nucleic acids” refers to nucleic acids not belonging to the host from which the sample was obtained. Non-host nucleic acids can be viral, bacterial, or other microbial DNA.
The term “microbial nucleic acids” refers to nucleic acids that is microbial (e.g., bacterial or viral or from a eukaryotic parasite) in origin that is present in a sample.
The term “host DNA” refers to DNA that originates from the multicellular eukaryotic subject from which the sample was obtained. Host DNA can be, e.g., plant or animal, including mammals particularly humans. The term “host DNA” includes nuclear DNA as well as DNA present in other organelles, e.g., the mitochondria and chloroplast (if the host is a plant), but not the DNA from microbes that often grow on or in the subject.
The term “non-host DNA” refers to DNA not belonging to the host from which the sample was obtained. Non-host DNA can be viral, bacterial, or other microbial DNA.
The term “microbial DNA” refers to genomic DNA that is microbial (e.g., bacterial or viral or from a eukaryotic parasite) in origin that is present in a sample.
The term “complementary DNA” or cDNA refers to a double-stranded DNA sample that was produced from an RNA sample by reverse transcription of RNA (using primers such as random hexamers or oligo-dT primers) followed by second-strand synthesis by digestion of the RNA with RNaseH and synthesis by DNA polymerase.
The term “RNA promoter adapter” is an adapter that contains a promoter for a bacteriophage RNA polymerase, e.g., the RNA polymerase from bacteriophage T3, T7, SP6 or the like.
The term “metagenomics sample” refers to a sample that contains more than one species of organism (eukaryotic, prokaryotic, or viral organisms).
The term “metagenomics analysis” refers to the analysis of a metagenomics sample.
Other definitions of terms may appear throughout the specification.
Objects of the Invention
In view of the problems associated with conventional methods and apparatus set forth above it is an object of the present invention to deplete selected nucleic acids from a nucleic acid sample using an enzyme mediated cutting mechanism.
It is another object of the present invention to provide a method that depletes selected host nucleic acids from sequencing libraries that include host and non-host nucleic acids in order to increase the proportion of non-host nucleic acid fragments for the purposes of pursuing downstream applications, including, but not limited to amplification, sequencing, cloning, etc.
It is another object of the present invention to provide a metagenomic analysis method that is unbiased relative to the fragments from across the genome of a particular species by selectively depleting some or all of the particular species' genome from a metagenomics sample.
It is further object of the present invention to gain better resolution in metagenomic analysis by increasing the number of sample reads that map to the a species' metagenome without increasing and in some instances while decreasing the amount of sequencing.
It is a further object of the present invention to provide methods and compositions to deplete mitochondrial DNA from a sample comprising mitochondrial DNA and non-mitochondrial DNA.
Nucleic Acids, Samples
Nucleic acids of the invention (targeted for enrichment, partitioning, or depletion) can be single stranded DNA, single stranded RNA, double stranded DNA, double stranded RNA, artificial DNA, artificial RNA, synthetic DNA, synthetic RNA, and RNA/DNA hybrids.
The nucleic acids of the invention can be a genomic fragment, comprising a region of the genome, or the whole genome itself. In one embodiment, the genome is a DNA genome. In another embodiment, the genome is a RNA genome.
Nucleic acids of the invention can be obtained from a eukaryotic or prokaryotic organism; from a mammalian organism or a non-mammalian organism; from an animal or a plant; from a bacteria or virus; from an animal parasite; or from a pathogen.
Nucleic acids of the invention can be obtained from any mammalian organism. In one embodiment the mammal is a human. In another embodiment the mammal is a livestock animal, for example a horse, a sheep, a cow, a pig, or a donkey. In another embodiment, a mammalian organism is a domestic pet, for example a cat, a dog, a gerbil, a mouse, a rat. In another embodiment the mammal is a type of a monkey.
Nucleic acids of the invention can be obtained from any bird or avian organism. An avian organism includes, but is not limited, to a chicken, turkey, duck and goose.
Nucleic acids of the invention can be obtained from a plant. In one embodiment, the plant is rice, maize, wheat, rose, grape, coffee, fruit, tomato, potato, or cotton.
In some embodiments, nucleic acids of the invention are obtained from a species of bacteria. In one embodiment, the bacteria are tuberculosis-causing bacteria.
In some embodiments, nucleic acids of the invention are obtained from a virus.
In some embodiments, nucleic acids of the invention are obtained from a species of fungi.
In some embodiments, nucleic acids of the invention are obtained from a species of algae.
In some embodiments, nucleic acids of the invention are obtained from any mammalian parasite.
In some embodiments, nucleic acids of the invention are obtained from any mammalian parasite. In one embodiment, the parasite is a worm. In another embodiment, the parasite is a malaria-causing parasite. In another embodiment, the parasite is a Leishmaniasis-causing parasite. In another embodiment, the parasite is an amoeba.
In one embodiment, the nucleic acids of the invention include nucleic acids that are targets of gRNAs (also referred to interchangeably herein as gRNA elements) and nucleic acids that are not the targets of the gRNAs, in the same sample.
In one embodiment, the nucleic acids in a sample include target nucleic acids/targeted sequences (targets of gRNAs) and nucleic acids of interest/sequences of interest (not targeted by gRNAs).
In one embodiment, the nucleic acids all belong to the same organism, but a subset is targeted for depletion or partitioning. For example nucleic acids of little informative value may be targeted. Examples of nucleic acids of little informative value include, but are not limited to: mitochondrial DNA, mitochondrial RNA, mitochondrial rRNA, repetitive sequence, multi-copy sequence, sequence encoding globin proteins, sequence encoding a transposon, sequence encoding retroviral sequence, sequence comprising telomere sequence, sequence comprising sub-telomeric repeats, sequence comprising centromeric sequence, sequence comprising intron sequence, sequence comprising Alu repeats, SINE repeats, LINE repeats, dinucleic acid repeats, trinucleic acid repeats, tetranucleic acid repeats, poly-A repeats, poly-T repeats, poly-C repeats, poly-G repeats, AT-rich sequence, or GC-rich sequence.
In one embodiment, a sample is contacted with CRISPR/Cas system protein-gRNA complexes, wherein the gRNAs are complementary to target sequences for depletion, the target sequences encoding globin proteins, sequences encoding a transposon, sequences encoding retroviral sequences, sequences comprising telomere sequences, sequences comprising sub-telomeric repeats, sequences comprising centromeric sequences, sequences comprising intron sequences, sequences comprising Alu repeats, sequences comprising SINE repeats, sequences comprising LINE repeats, sequences comprising dinucleic acid repeats, sequences comprising trinucleic acid repeats, sequences comprising tetranucleic acid repeats, sequences comprising poly-A repeats, sequences comprising poly-T repeats, sequences comprising poly-C repeats, sequences comprising poly-G repeats, sequences comprising AT-rich sequences, or sequences comprising GC-rich sequences.
In one exemplary embodiment, ribosomal RNA may be targeted for depletion or partitioning. In another exemplary embodiment repetitive DNA may be targeted for depletion or partitioning. In such an embodiment, the present method is usable to deplete the ribosomal or repetitive DNA or any other DNA that has little informative value in order to reduce the cost of sequencing these elements of the DNA extraction and improve data yield.
In one embodiment, the target DNA may be non-mitochondrial DNA (e.g. genomic DNA), and the DNA of interest may be the mitochondrial DNA, and the mitochondrial DNA is enriched by targeting and depleting the non-mitochondrial human DNA.
In one embodiment, the target DNA for depletion may be mitochondrial DNA, and the DNA of interest may be the non-mitochondrial DNA, and the non-mitochondrial DNA is enriched by targeting and depleting the mitochondrial human DNA. In an exemplary embodiment, an ATAC-Seq procedure (for example see WO2014/189957 is carried out resulting in unwanted residual mitochondrial DNA; the methods of the invention can be used to deplete unwanted mitochondrial DNA from the sample.
In one embodiment, the nucleic acids to be depleted or partitioned out may be a non-mappable region of a genome; and the nucleic acids to be retained for further analysis/sequencing/cloning may be mappable regions of a genome. In one embodiment, the nucleic acids to be depleted or partitioned out may be a mappable region of a genome; and the nucleic acids to be retained for further analysis/sequencing/cloning may be non-mappable regions of a genome. Examples of non mappable regions include telomeres, centromeres, repetitive regions, or other genomic regions that contain features harder to map.
In some embodiments, the methods of the invention are carried out on samples comprising host nucleic acids and non-host nucleic acids. In one embodiment, the host nucleic acids are mammalian, and the non-host nucleic acids are not mammalian (e.g., bacterial, viral, fungal, protozoan). In one embodiment, the host nucleic acids are human and the non-host nucleic acids are bacterial. In one embodiment, the host nucleic acids are human and the non-host nucleic acids are viral. In one embodiment, the host nucleic acids are human and the non-host nucleic acids are fungal. In one embodiment, the host nucleic acids are human and the non-host nucleic acids are protozoan. In one embodiment, the host nucleic acids are from a type of livestock. In one embodiment, the host nucleic acids are from a monkey. In one specific embodiment, the host nucleic acids are human and the non-host nucleic acids are from an unidentified pathogen, for example a known or unknown virus, or a known or unknown bacteria, or a known or unknown animal parasite. In one specific embodiment, the host nucleic acids are from a cow and the non-host nucleic acids are viral, bacterial, fungal, or protozoan.
In samples that contain both host and non-host nucleic acids, the host and non-host may have, for example, a host-pathogen relationship or a symbiotic relationship. In some embodiments, the non-host fraction of a total nucleic acid sample is obtained from a host may be derived from the microbiome that is associated with the host.
In one embodiment, a sample obtained from a host organism (e.g. a human host) contains nucleic acids from more than one non-host organism, e.g. from a plurality of non-host organisms comprising at least one unknown non-host organism to be identified. The compositions and methods of the invention can be utilized to serially process the nucleic acids in the sample to first deplete/partition out the host-DNA, and then subsequently deplete/partition out other known non-host nucleic acids that are not of interest, to arrive at a remaining pool of nucleic acids that represent a particular non-host of interest. For example, such an embodiment would be applicable in a situation where it a host would be expected to be harboring more than one known bacteria, for example existing symbiotically. For example, such an embodiment would be applicable to detect an unknown pathogen in a saliva sample from an individual, the saliva being known to harbor several species of bacteria, for example species of Streptococcus. In another example, the sample may be a fecal sample from a mammalian host, comprising known and unknown non-host nucleic acids.
In one embodiment, a sample obtained from a known host organism (e.g., a human host) contains non-host nucleic acids but the non-host is not known (e.g. an unknown pathogenic bacteria, virus, or other pathogen in the human blood) until the host nucleic acids are depleted or partitioned, such that the non-host nucleic acids are enriched and be subjected to further downstream analysis.
In an exemplary embodiment, where the DNA includes host-DNA and non-host DNA the present method is usable to deplete substantially all of the host DNA in order to build a library of non-host DNA for further analysis. In cases where the DNA includes nucleic and mitochondrial DNA the present method is usable to deplete the mitochondrial DNA in order to build a library of nuclear DNA for further analysis; or to deplete the nuclear DNA in order to build a library of mitochondrial DNA for further analysis. In cases where the DNA includes abundant sequences of ribosomal RNA or repetitive DNA that may have little informative value, the present method is usable to deplete the ribosomal or repetitive DNA or any other DNA that has little informative value in order to reduce the cost of sequencing these elements of the DNA extraction and improve data yield. In each application, the resulting DNA has significantly fewer DNA fragments to sequence thereby reducing sequencing cost and complexity. Moreover according to the methods of the present invention the depleted sample still provides all of the DNA material present in the biological sample that was not actively depleted which provides the advantage of reducing the sequencing costs as well as improving the data yield by sequencing smaller less diverse DNA samples.
In one embodiment, the nucleic acids of the invention are obtained from a biological sample. The biological sample from which the nucleic acids are obtained include but are not limited to whole blood, plasma, serum, tears, saliva, mucous, cerebrospinal fluid, teeth, bone, fingernails, feces, urine, tissue, biopsy, etc. The biological sample may include forensic samples such as teeth, bone, fingernails or the like. The biological sample may include tissue, a tissue biopsy, for example a resected lung tissue. The biological sample may include a clinical sample, which refers to a sample obtained in a clinical setting, such as a in a hospital, or clinic.
In one embodiment, the nucleic acids of the invention are obtained from an environmental sample, for example from water, soil, air, or rock.
In one embodiment, the nucleic acids of the invention are obtained from a forensic sample, for example, a sample obtained from an individual at a crime scene, from a piece of evidence, post-mortem, as a part of an ongoing investigation or the like.
In on embodiment, the nucleic acids of the invention are provided in a library.
The nucleic acids of the invention are either provided or extracted from a sample. Extraction can extract substantially all the nucleic acid sequences from a specimen. This can be, for example, nucleic acid sequences of the host and nucleic acid sequences of any non-human or non-host organism present in the specimen.
An extraction may produce host nucleic acids and non-host nucleic acids at a ratio of anywhere between 99.999:0.001 to 0.001:99.999. An extraction may produce targeted nucleic acids and nucleic acids of interest at a ratio of anywhere between 99.999:0.001 to 0.001:99.999. An extraction may produce nucleic acids to be depleted to nucleic acids to be retained/analyzed/sequenced at a ratio of anywhere between 99.999:0.001 to 0.001:99.999. An extraction may produce nucleic acids to be partitioned to nucleic acids to be retained/analyzed/sequenced at a ratio of anywhere between 99.999:0.001 to 0.001:99.999. An extraction from a biological sample may produce host nucleic acids to be depleted to nucleic acids from more than one non-host, to be retained/analyzed/sequenced, at a ratio of anywhere between 99.999:0.001 to 0.001:99.999. In these embodiments, the ratios can be equal to or fall anywhere in between 99.999:0.001 to 0.001:99.999, for example the ratio can be 99:1, 95:5, 90:10, 85:15, 80:20, 75:25, 70: 30, 65:35, 60:40, 55:45, 50:50, 45:55, 40:60, 35:65, 30:70, 25:75, 20:80, 15:85, 10:90, 5:95, and 1:99.
After the extraction, the extracted nucleic sequences can be fragmented to reduce the lengths of each extracted nucleic acids to a more manageable length for amplifying, sequencing or the like.
As provided herein, depletion of at least 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90% of the starting nucleic acid material can be depleted or partitioned. This depletion or partitioning can be achieved in no greater than 10 minutes, 15 minutes, 20 minutes, 30 minutes, 45 minutes, 60 minutes, 75 minutes, 90 minutes, 105 minutes, 120 minutes, 150 minutes, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours or 12 hours.
In one embodiment, the nucleic acids of the invention are adapter-ligated. Nucleic acids of the invention to be adapter-ligated can range from 10 bp to 1000 bp. For example the nucleic acid to be adapter-ligated may be at least 10, 15, 20, 25, 50, 75, 100, 125, 150, 175, 200, 25, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 bp. In one specific embodiment, the nucleic acid to be adapter ligated is 100 bp. In one specific embodiment, the nucleic acid to be adapter ligated is 200 bp. In one specific embodiment, the nucleic acid to be adapter ligated is 300 bp. In one specific embodiment, the nucleic acid to be adapter ligated is 400 bp. In one specific embodiment, the nucleic acid to be adapter ligated is 500 bp.
In some embodiments a sample comprises a library of nucleic acid fragments, wherein the nucleic acid fragments are adapter-ligated on their 5′ and 3′ ends. In such embodiments an adapter is ligated to each end of each of the nucleic acid fragments, at the 5′ and 3′ ends. In other embodiments an adapter may be ligated to only one end of each of the fragments. In some embodiments, adapters further comprise intervening sequence between the 5′ terminal end and/or the 3′ terminal end. For example an adapter can further comprise a barcode sequence.
In some embodiments the adapter is a nucleic acid that is ligatable to both strands of a double-stranded DNA molecule.
In some embodiments, adapters are ligated prior to depletion/enrichment. In other embodiments, adapters are ligated at a later step.
In some embodiments the adapters are linear. In some embodiments the adapters are linear Y-shaped. In some embodiments the adapters are linear circular. In some embodiments the adapters are hairpin adapters.
In various embodiments the adapter may be a hairpin adapter i.e., one molecule that base pairs with itself to form a structure that has a double-stranded stem and a loop, where the 3′ and 5′ ends of the molecule ligate to the 5′ and 3′ ends of the double-stranded DNA molecule of the fragment, respectively.
Alternately, the adapter may be a Y-adapter ligated to one end or to both ends of a fragment, also called a universal adapter. Alternately, the adapter may itself be composed of two distinct oligonucleotide molecules that are base paired with one another. Additionally a ligatable end of the adapter may be designed to be compatible with overhangs made by cleavage by a restriction enzyme, or it may have blunt ends or a 5′ T overhang.
The adapter may include double-stranded as well as single-stranded molecules. Thus the adapter can be DNA or RNA, or a mixture of the two. Adapters containing RNA may be cleavable by RNase treatment or by alkaline hydrolysis.
Adapters can be 10 to 100 bp in length although adapters outside of this range are usable without deviating from the present invention. In specific embodiments, the adapter is at least 10 bp, at least 15 bp, at least 20 bp, at least 25 bp, at least 30 bp, at least 35 bp, at least 40 bp, at least 45 bp, at least 50 bp, at least 55 bp, at least 60 bp, at least 65 bp, at least 70 bp, at least 75 bp, at least 80 bp, at least 85 bp, at least 90 bp, or at least 95 bp in length.
In certain cases, an adapter may comprise an oligonucleotide designed to match a nucleotide sequence of a particular region of the host genome, e.g., a chromosomal region whose sequence is deposited at NCBI's Genbank database or other databases. Such an oligonucleotide may be employed in an assay that uses a sample containing a test genome, where the test genome contains a binding site for the oligonucleotide. In further examples the fragmented nucleic acid sequences may be derived from one or more DNA sequencing libraries. An adapter may be configured for a next generation sequencing platform, for example for use on an Illumina sequencing platform or for use on an IonTorrents platform, or for use with Nanopore technology.
Guide RNAs
Provided herein are guide RNAs (gRNAs), wherein the gRNAs are complementary to (selective for, can hybridize with) targeted sites or targeted sequences in the nucleic acids, for example in genomic DNA from a host. In one embodiment, the present invention provides a guide RNA library which comprises a collection of gRNAs, configured to hybridize with a nucleic acid sequence targeted for depletion or partitioning.
In one embodiment, the gRNAs are selective for target nucleic acids (or targeted sequences) in a sample, but are not selective for sequences of interest in the sample.
In one embodiment, the gRNAs are selective for host nucleic acids in a biological sample from a host, but are not selective for non-host nucleic acids in the sample from a host. In one embodiment, the gRNAs are selective for non-host nucleic acids from a biological sample from a host but are not selective for the host nucleic acids in the sample. In one embodiment, the gRNAs are selective for both host nucleic acids and a subset of the non-host nucleic acids in a biological sample from a host. For example, where a complex biological sample comprises host nucleic acids and nucleic acids from more than one non-host organisms, the gRNAs may be selective for more than one of the non-host species. In such embodiments, the gRNAs are used to serially deplete or partition the sequences that are not of interest. For example, saliva from a human contains human DNA, as well as the DNA of more than one bacterial species, but may also contain the genomic material of an unknown pathogenic organism. In such an embodiment, gRNAs directed at the human DNA and the known bacteria can be used to serially deplete the human DNA, and the DNA of the known bacterial, thus resulting in a sample comprising the genomic material of the unknown pathogenic organism.
In an exemplary embodiment, the gRNAs are selective for human host DNA obtained from a biological sample from the host, but do not hybridize with DNA from an unknown organism (e.g. pathogen(s)) also in the sample.
In some embodiments, the gRNAs are selective for a target nucleic acid sequences which are followed by Protospacer Adjacent Motif (PAM) sequences that can be bound by a Cas9. In some embodiments, the sequence of the gRNAs is determined by the CRISPR/Cas system protein type. For example, in various embodiments the gRNAs may be tailored to different Cas9 types as the PAM sequence can vary by the species of the bacteria from which Cas9 is derived.
gRNAs can range in size for example, from 50-250 base pairs. For example a gRNA can be at least 50 bp, 55 bp, 60 bp, 65 bp, 70 bp, 75 bp, 80 bp, 85 bp, 90 bp, 95 bp, 100 bp, 110 bp, 120 bp, 125 bp, 130 bp, 140 bp, 150 bp, 160 bp, 170 bp, 175 bp, 180 bp, 190 bp, or 195 bp. In specific embodiments, the gRNA is 80 bp, 90 bp, 100 bp, or 110 bp. Each target-specific gRNA comprises a base pair sequence that is complementary to a pre-defined site in a target nucleic acid that is followed by a Protospacer Adjacent Motif or (PAM) sequence that can be bound by a CRISPR/Cas system protein, for example a Cas9 protein, derived from a bacterial species. In specific embodiments, the base pair sequence of the gRNA that is complementary to a pre-defined site in a target nucleic acid is 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 base pairs.
The present invention also provides for gRNA libraries. A gRNA library can comprise a number of different species-specific gRNAs each, configured to hybridize with (be selective for) a nucleic acid sequence being targeted for depletion or partitioning. Each gRNA includes a target-specific guide sequence and a stem loop binding site that is formed to bind with a particular CRISPR/Cas system protein, for example with a Cas9 protein. The library can comprise a plurality of different guide RNAs, each having a different 15 to 20 base pair sequence that is complementary to a different pre-defined site in the nucleic acid being targeted, that is followed by an appropriate PAM sequence that can be bound by a CRISPR/Cas system protein. For each guide RNA the PAM sequence is present in the pre-defined DNA target sequence of the nucleic acid of interest but is not present in the corresponding target specific guide sequence.
Generally according to the present invention, any nucleic acid sequence in a genome of interest, with a pre-defined target sequence followed by the appropriate PAM sequence can be hybridized by a corresponding guide RNA provided in the guide RNA library and bound by CRISPR/Cas system protein, for example Cas9. In various embodiments the gRNA library may be tailored to different CRISPR/Cas system proteins, for example different Cas9 types since the PAM sequence can vary by the species of the bacteria from which protein is derived.
Different target specific sequences in the gRNAs can be generated. This can be done by using a promoter for a bacteriophage RNA polymerase, e.g., the RNA polymerase from bacteriophage T3, T7, SP6 or the like. Accordingly each different T7 RNA polymerase promoter provides a different target specific sequence suitable for hybridizing to a different target nucleic acid sequence. A non-limiting exemplary set of forward primers usable for both annealing and subsequent PCR reactions is listed in Tables 1 and 2 of Example 1 provided below.
Without being limited to theory, the distance between gRNAs to arrive at >95% cleavage of the target nucleic acid can be computed, if the gRNAs display ˜100% efficacy: this can be computed by measuring the distribution of library size and determining the mean, N and the standard deviation SD; N-2SD=minimum size for >95% of the library, ensuring that there is one guide RNA per fragment of this size to ensure >95% depletion. This can also be described as the Maximum distance between guide RNAs=Mean of library size−2× (standard deviation of library size).
In the embodiments provided herein a gRNA library can be amplified to include a large number of copies of each different gRNAas well as a large number of different gRNAs as may be suitable to for the desired depletion results. The number of unique gRNAs in a given guide RNA library may range from 1 unique gRNAs to as many as 1010 unique gRNAs or approximately 1 unique guide RNA sequence for every 2 base pairs in the human genome. In some embodiments, the library comprises, at least 102 unique gRNAs. In some embodiments library comprises at least 102, at least 103, at least 104, at least 105, at least 106, at least 107, at least 108, at least 109, at least 1010 unique gRNAs. In one exemplary embodiment, the library comprises about 103 unique gRNAs.
In the embodiments provided herein, the methods comprise contacting a sample comprising nucleic acids with a plurality of CRISPR/Cas9 system protein-gRNA complexes, wherein the gRNAs are complementary to target sequences, such sequences targeted for depletion. In some embodiments, the method comprises using gRNAs which can base-pair with the targeted sites, wherein the sample is contacted with at least 102 unique gRNAs. In some embodiments the sample is contacted with at least 102, at least 103, at least 104, at least 105, at least 106, at least 107, at least 108, at least 109, at least 1010 unique gRNAs. In one exemplary embodiment, the sample is contacted with about 103 unique gRNAs.
In the embodiments provided herein, the methods comprise contacting a sample with at least 102 unique CRISPR/Cas system protein-gRNA complexes, where the unique nature of the complex is determined by the unique nature of the gRNA itself. For example, 2 unique CRISPR/Cas system protein-gRNA complexes may share the same CRISPR/Cas system protein, but the gRNAs differ, even if by only 1 nucleotide. Thus, in some embodiments, the method comprises contacting a sample with at least 102 unique CRISPR/Cas system protein-gRNA complexes. In some embodiments the sample is contacted with at least 102, at least 103, at least 104, at least 105, at least 106, at least 107, at least 108, at least 109, at least 1010 unique CRISPR/Cas system protein-gRNA complexes. In one exemplary embodiment, the sample is contacted with about 103 unique CRISPR/Cas system protein-gRNA complexes.
In the embodiments provided herein, the methods comprise contacting a sample comprising a genomic DNA with a plurality of CRISPR/Cas9 system protein-gRNA complexes, wherein the gRNAs are complementary to sites targeted in the genome for depletion. In some embodiments, the method comprises using gRNAs which can base-pair with the targeted DNA, wherein the target site of interest is spaced at least every 1 bp, at least every 2 bp, at least every 3 bp, at least every 4 bp, at least every 5 bp, at least every 6 bp, at least every 7 bp, at least every 8 bp, at least every 9 bp, at least every 10 bp, at least every 11 bp, at least every 12 bp, at least every 13 bp, at least every 14 bp, at least every 15 bp, at least every 16 bp, at least every 17 bp, at least every 18 bp, at least every 19 bp, 20 bp, at least every 25 bp, at least every 30 bp, at least every 40 bp, at least every 50 bp, at least every 100 bp, at least every 200 bp, at least every 300 bp, at least every 400 bp, at least every 500 bp, at least every 600 bp, at least every 700 bp, at least every 800 bp, at least every 900 bp, at least every 1000 bp, at least every 2500 bp, at least every 5000 bp, at least every 10,000 bp, at least every 15,000 bp, at least every 20,000 bp, at least every 25,000 bp, at least every 50,000 bp, at least every 100,000 bp, at least every 250,000 bp, at least every 500,000 bp, at least every 750,000 bp, or even at least every 1,000,000 bp across a genome of interest.
In the embodiments provided herein, the methods comprise contacting a sample comprising nucleic acids targeted for depletion with a plurality of CRISPR/Cas9 system protein-gRNA complexes, wherein the gRNAs are complementary to the nucleic acids targeted for depletion. In some embodiments the molar ratio of the gRNA:nucleic acids targeted for depletion is 1:1, 5:1, 10:1, 50:1, 100:1, 150:1, 250:1,500:1,750:1,1000:1, 1500:1, 2000:1, 2500:1, 5000:1, 7500:1, or even 10,000:1. In an exemplary embodiment the molar ratio of the gRNA:nucleic acids targeted for depletion is 500:1.
In the embodiments provided herein, the methods comprise contacting a sample comprising nucleic acids targeted for depletion with a plurality of CRISPR/Cas9 system protein-gRNA complexes, wherein the gRNAs are complementary to the nucleic acids targeted for depletion. In some embodiments the weight to weight ratio of the gRNA:nucleic acids targeted for depletion is 1:1, 5:1, 10:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, 100:1, 150:1, 250:1,500:1,750:1,1000:1, 1500:1, 2000:1, 2500:1, 5000:1, 7500:1, or even 10,000:1. In an exemplary embodiment the weight to weight ratio of the gRNA:nucleic acids targeted for depletion is ranges between 50:1 and 100:1.
The following description refers to
In
In
CRISPR/Cas System Proteins
Provided herein compositions and methods for the depletion of unwanted nucleic acids, and/or enrichment of sequences of interest in a sample. These compositions and methods utilize a CRISPR/Cas system protein.
In some embodiments, CRISPR/Cas system proteins include proteins from CRISPR Type I systems, CRISPR Type II systems, and CRISPR Type III systems.
In some embodiments, CRISPR/Cas system proteins can be from any bacterial or archaeal species.
In some embodiments, the CRIPR/Cas system proteins are from, or are derived from CRISPR/Cas system proteins from Streptococcus pyogenes, Staphylococcus aureus, Neisseria meningitidis, Streptococcus thermophiles, Treponema denticola, Francisella tularensis, Pasteurella multocida, Campylobacter jejuni, Campylobacter lari, Mycoplasma gallisepticum, Nitratifractor salsuginis, Parvibaculum lavamentivorans, Roseburia intestinalis, Neisseria cinerea, Gluconacetobacter diazotrophicus, Azospirillum, Sphaerochaeta globus, Flavobacterium columnare, Fluviicola taffensis, Bacteroides coprophilus, Mycoplasma mobile, Lactobacillus farciminis, Streptococcus pasteurianus, Lactobacillus johnsonii, Staphylococcus pseudintermedius, Filifactor alocis, Legionella pneumophila, Suterella wadsworthensis, or Corynebacter diphtheria.
In some embodiments, examples of CRISPR/Cas system proteins can be naturally occurring or engineered versions.
In some embodiments, naturally occurring CRISPR/Cas system proteins include Cas9, Cpf1, Cas3, Cas8a-c, Cas10, Cse1, Csy1, Csn2, Cas4, Csm2, and Cm5. In an exemplary embodiment, the CRISPR/Cas system protein comprises Cas9.
A “CRISPR/Cas system protein-gRNA complex” refers to a complex comprising a CRISPR/Cas system protein and a guide RNA. The guide RNA may be composed of two molecules, i.e., one RNA (“crRNA”) which hybridizes to a target and provides sequence specificity, and one RNA, the “tracrRNA”, which is capable of hybridizing to the crRNA. Alternatively, the guide RNA may be a single molecule (i.e., a gRNA) that contains crRNA and tracrRNA sequences. A CRISPR/Cas system protein may be at least 60% identical (e.g., at least 70%, at least 80%, or 90% identical, at least 95% identical or at least 98% identical or at least 99% identical) to a wild type CRISPR/Cas system protein. The CRISPR/Cas system protein may have all the functions of a wild type CRISPR/Cas system protein, or only one or some of the functions, including binding activity, nuclease activity, and nuclease activity.
The term “CRISPR/Cas system protein-associated guide RNA” refers to a guide RNA as described above (comprising a crRNA molecule and a tracrRNA molecule, or comprising a single RNA molecule that includes both crRNA and tracrRNA sequences). The CRISPR/Cas system protein-associated guide RNA may exist as isolated RNA, or as part of a CRISPR/Cas system protein-gRNA complex.
Cas9
In some embodiments, the CRISPR/Cas system protein comprises Cas9. The Cas9 of the present invention can be isolated, recombinantly produced, or synthetic.
Cas9 proteins that can be used in the embodiments herein can be found in http://www.nature.com/nature/journal/v520/n7546/full/nature14299.html.
In some embodiments, the Cas9 is a Type II CRISPR system derived from Streptococcus pyogenes, Staphylococcus aureus, Neisseria meningitidis, Streptococcus thermophiles, Treponema denticola, Francisella tularensis, Pasteurella multocida, Campylobacter jejuni, Campylobacter lari, Mycoplasma gallisepticum, Nitratifractor salsuginis, Parvibaculum lavamentivorans, Roseburia intestinalis, Neisseria cinerea, Gluconacetobacter diazotrophicus, Azospirillum, Sphaerochaeta globus, Flavobacterium columnare, Fluviicola taffensis, Bacteroides coprophilus, Mycoplasma mobile, Lactobacillus farciminis, Streptococcus pasteurianus, Lactobacillus johnsonii, Staphylococcus pseudintermedius, Filifactor alocis, Legionella pneumophila, Suterella wadsworthensis, or Corynebacter diphtheria.
In some embodiments, the Cas9 is a Type II CRISPR system derived from S. pyogenes and the PAM sequence is NGG located on the immediate 3′ end of the target specific guide sequence. The PAM sequences of Type II CRISPR systems from exemplary bacterial species can also include: Streptococcus pyogenes (NGG), Staph aureus (NNGRRT), Neisseria meningitidis (NNNNGA TT), Streptococcus thermophiles (NNAGAA) and Treponema denticola (NAAAAC) which are all usable without deviating from the present invention. ((http://www.nature.com/nature/journal/v520/n7546/full/nature14299.html)).
In one exemplary embodiment, Cas9 sequence can be obtained, for example, from the pX330 plasmid (available from Addgene), re-amplified by PCR then cloned into pET30 (from EMD biosciences) to express in bacteria and purify the recombinant 6His tagged protein.
The “Cas9-gRNA complex” refers to a complex comprising a Cas9 protein and a guide RNA. The guide RNA may be composed of two molecules, i.e., one RNA (“crRNA”) which hybridizes to a target and provides sequence specificity, and one RNA, the “tracrRNA”, which is capable of hybridizing to the crRNA. Alternatively, the guide RNA may be a single molecule (i.e., a gRNA) that contains crRNA and tracrRNA sequences. A Cas9 protein may be at least 60% identical (e.g., at least 70%, at least 80%, or 90% identical, at least 95% identical or at least 98% identical or at least 99% identical) to a wild type Cas9 protein, e.g., to the Streptococcus pyogenes Cas9 protein. The Cas9 protein may have all the functions of a wild type Cas9 protein, or only one or some of the functions, including binding activity, nuclease activity, and nuclease activity.
The term “Cas9-associated guide RNA” refers to a guide RNA as described above (comprising a crRNA molecule and a tracrRNA molecule, or comprising an RNA molecule that includes both crRNA and tracrRNA sequences). The Cas9-associated guide RNA may exist as isolated RNA, or as part of a Cas9-gRNA complex.
Catalytically Dead CRISPR/Cas System Proteins
In some embodiments, engineered examples of CRISPR/Cas system proteins include catalytically dead CRISPR/Cas system proteins. The term “catalytically dead” generally refers to a CRISPR/Cas system protein that has inactivated HNH and RuvC nucleases. Such a protein can bind to a target site in any nucleic acid (where the target site is determined by the guide RNA), but the protein is unable to cleave or nick the double-stranded DNA.
In some embodiments, the catalytically dead CRISPR/Cas system protein is dCas9, dCpf1, dCas3, dCas8a-c, dCas10, dCse1, dCsy1, dCsn2, dCas4, dCsm2, or dCm5.
In one embodiment the catalytically dead CRISPR/Cas system protein is a dCas9. Accordingly the dCas9 allows partition of the mixture into unbound nucleic acids and dCas9-bound fragments targeted for partitioning. In one embodiment, a dCas9/gRNA complex binds to targets determined by the gRNA sequence. The dCas9 bound can prevent cutting by Cas9 while other manipulations proceed. This is depicted in
CRISPR/Cas System Protein Nickases
In some embodiments, engineered examples of CRISPR/Cas system proteins also include Cas nickases. A Cas nickase refers to a modified version of a CRISPR/Cas system protein, containing a single inactive catalytic domain.
In some embodiments, the CRISPR/Cas system protein nickase is Cas9 nickase, Cpf1 nickase, Cas3 nickase, Cas8a-c nickase, Cas10 nickase, Cse1 nickase, Csy1 nickase, Csn2 nickase, Cas4 nickase, Csm2 nickase, or Cm5 nickase.
In one embodiment, the CRISPR/Cas system protein nickase is Cas9 nickase.
In some embodiments, a Cas9 nickase can be used to bind to target sequence. The term “Cas9 nickase” refers to a modified version of the Cas9 protein, containing a single inactive catalytic domain, for example, either the RuvC- or the HNH-domain. With only one active nuclease domain, the Cas9 nickase cuts only one strand of the target DNA, creating a single-strand break or “nick”. Depending on which mutant is used, the guide RNA-hybridized strand or the non-hybridized strand may be cleaved. Cas9 nickases bound to 2 gRNAs that target opposite strands can create a double-strand break in the DNA. This “dual nickase” strategy increases the specificity of cutting because it requires that both Cas9/gRNA complexes be specifically bound at a site before a double-strand break is formed.
In an exemplary embodiment, depletion of DNA can be carried out using a Cas9 nickase. In one embodiment, the method comprises: making a DNA sequencing library comprising DNA to be removed (for example human DNA not of interest) and DNA of interest (for example an DNA from an unknown pathogen); designing guide RNAs so that all the DNA to be depleted will have two guide RNA binding sites in close proximity (for example, less than 15 bases apart) on opposite DNA strands; adding Cas9 Nickase and guide RNA to the DNA library. In this embodiment, the Cas9 Nickase recognizes its target sites on the DNA to be removed and cuts only one strand. For DNA to be depleted, two separate Cas9 Nickases can cut both strands of the DNA to be removed (e.g. human DNA) in close proximity; only the DNA to be removed (e.g. human DNA) will have two Cas9 nickase sites in close proximity which creates a double stranded break. If a Cas9 Nickase recognizes non-specifically or at low affinity a site on the DNA of interest (e.g. pathogen DNA), it will only cut one strand which would not prevent subsequent PCR amplification or downstream processing of the DNA molecule. This is pictorially depicted in
Dissociable and Thermostable CRISPR/Cas System Proteins
Although CRISPR/Cas System proteins can be used in combination with a library of guide RNAs to efficiently deplete a collection of target DNA, large amounts (e.g. >30 pmoles) of CRISPR/Cas System proteins and guide RNAs may be needed. One reason for this usually >100 fold excess amount over target DNA is that, unlike classical restriction enzymes such as EcoRI, which detach completely from their target DNA after cleavage, CRISPR/Cas System proteins are not completely recycled after completion of the cutting reaction. CRISPR/Cas System proteins, for example Cas9, can remain bound to one of the two daughter DNA product molecules (see
In some embodiments, to overcome this problem, dissociable CRISPR/Cas System proteins are provided herein. For example upon cleavage of targeted sequences, the CRISPR/Cas System protein can be made to dissociate from the gRNA, or from the target. In some embodiments, the dissociation is induced by elevating the temperature of the reaction. This can act to increase processivity of the enzyme, by allowing it to complex with available gRNAs, re-associate with additional target sequences and generate additional cleaved target sequences.
In some embodiments to overcome this problem, thermostable CRISPR/Cas System proteins are provided herein. In such embodiments, the reaction temperature is elevated, inducing dissociation of the protein; the reaction temperature is lowered, allowing for the generation of additional cleaved target sequences. In some embodiments, thermostable CRISPR/Cas system proteins maintain at least 50% activity, at least 55% activity, at least 60% activity, at least 65% activity, at least 70% activity, at least 75% activity, at least 80% activity, at least 85% activity, at least 90% activity, at least 95% activity, at least 96% activity, at least 97% activity, at least 98% activity, at least 99% activity, or 100% activity, when maintained for at least 75° C. for at least 1 minute. In some embodiments, thermostable CRISPR/Cas system proteins maintain at least 50% activity, when maintained for at least 1 minute at least at 75° C., at least at 80° C., at least at 85° C., at least at 90° C., at least at 91° C., at least at 92° C., at least at 93° C., at least at 94° C., at least at 95° C., 96° C., at least at 97° C., at least at 98° C., at least at 99° C., or at least at 100° C. In some embodiments, thermostable CRISPR/Cas system proteins maintain at least 50% activity, when maintained at least at 75° C. for at least 1 minute, 2 minutes, 3 minutes, 4 minutes, or 5 minutes. In some embodiments, a thermostable CRISPR/Cas system protein maintains at least 50% activity when the temperature is elevated, lowered to 25° C.-50° C. In some embodiments, the temperature is lowered to 25° C., to 30° C., to 35° C., to 40° C., to 45° C., or to 50° C. In one exemplary embodiment, a thermostable enzyme retains at least 90% activity after 1 min at 95° C.
In some embodiments, the thermostable CRISPR/Cas system protein is thermostable Cas9, thermostable Cpf1, thermostable Cas3, thermostable Cas8a-c, thermostable Cas10, thermostable Cse1, thermostable Csy1, thermostable Csn2, thermostable Cas4, thermostable Csm2, or thermostable Cm5.
In some embodiments the thermostable CRISPR/Cas system protein is thermostable Cas9.
In one exemplary embodiment, thermostable Cas9 complexed with a guide RNAs (for example a gRNA library against human DNA) can be applied to a sequencing library of DNA mixture (containing for example 95% human DNA and 5% viral DNA). As depicted in
Thermostable CRISPR/Cas System proteins can be isolated, for example, identified by sequence homology in the genome of thermophilic bacteria Streptococcus thermophilus and Pyrococcus furiosus. CRISPR/Cas system genes can then be cloned into an expression vector. In one exemplary embodiment, a thermostable Cas9 protein is isolated.
In another embodiment, a thermostable CRISPR/Cas system protein can be obtained by in vitro evolution of a non-thermostable CRISPR/Cas system protein. The sequence of a CRISPR/Cas system protein can be mutagenized to improve its thermostability. In some embodiments, this can be achieved by site-directed mutagenesis to remove excess loop sequences, increasing the number of ionic bridges between protein domains, or by diluting into droplets and PCR to create a pool of potential mutants. In one exemplary embodiment, a thermostable Cas9 is produced by in vitro evolution of a non-thermostable Cas9.
Methods of the Invention
Enrichment of Sequences of Interest/Depletion of Targeted Sequences
Provided herein are methods of enriching a sample for sequences of interest, by depleting targeted sequences that are not of interested (target sequences are sequences targeted for depletion).
In one embodiment, a method of enriching a sample for sequences of interest, comprises:
providing a sample comprising sequences of interest and targeted sequences for depletion, wherein the sequences of interest comprise less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, less than 0.1%, less than 0.05%, less than 0.01%, less than 0.005%, or even less than 0.001% of the sample; contacting the sample with a plurality of CRISPR/Cas system protein-gRNA complexes, wherein the gRNAs are complementary to the targeted sequences, whereby the targeted sequences are cleaved. In an exemplary embodiment the CRISPR/Cas system protein is Cas9.
In one embodiment, a method of enriching a sample for non-mitochondrial DNA comprises: (a) providing a sample comprising mitochondrial DNA and non-mitochondrial DNA, wherein the mitochondrial DNA and non-mitochondrial DNA are adapter-ligated, and wherein the adapters are ligated to the 5′ and 3′ ends of the mitochondrial DNA and non-mitochondrial DNA; (b) contacting the sample with a plurality of CRISPR/Cas system protein-gRNA complexes, wherein the gRNAs are complementary to the mitochondrial DNA, thereby generating mitochondrial DNA adapter ligated only on one end and non-mitochondrial DNA adapter ligated on both the 5′ and 3′ ends; and (c) enriching the sample for non-mitochondrial DNA. In some embodiments the sample is enriched using adapter-specific PCR. In some embodiments, the enriching comprises treating the product of step (b) with an enzyme that has exonuclease activity, for example Exonuclease III or BAL-31. For example, if Exonuclease III is utilized, the adapters may be Y-shaped. If BAL-31 is utilized, the adapters may comprise poly-G tails. In some embodiments the sample is enriched by purification of the sequences of interest.
In another embodiment, a method of enriching a sample comprises: (a) providing a sample comprising nucleic acids from a first genome and nucleic acids from a second genome, wherein the nucleic acids from the first genome are adapter-ligated on their 5′ and 3′ ends and wherein the nucleic acids from the second genome are adapter-ligated on their 5′ and 3′ ends; (b) contacting the sample with a plurality of CRISPR/Cas system protein-gRNA complexes, wherein the gRNAs are complementary to sites on the nucleic acids from the first genome, thereby generating nucleic acids from the first genome adapter ligated only on one end and nucleic acids from the second genome adapter ligated on both the 5′ and 3′ ends; and (c) enriching the sample for nucleic acids from the first genome. In some embodiments the sample is enriched using adapter-specific PCR. In some embodiments, the enriching comprises treating the product of step (b) with an enzyme that has exonuclease activity, for example Exonuclease III or BAL-31. For example, if Exonuclease III is utilized, the adapters may be Y-shaped. If BAL-31 is utilized, the adapters may comprise poly-G tails. In some embodiments the sample is enriched by purification of the sequences of interest.
In another embodiment, a method of enriching a sample for non-host nucleic acids comprises: providing a sample comprising host nucleic acids and non-host nucleic acids, wherein the host nucleic acids and non-host nucleic acids are adapter-ligated, and wherein the adapters are ligated to the 5′ and 3′ ends of the host nucleic acids and the non-host nucleic acids; contacting the sample with a plurality of CRISPR/Cas system protein-gRNA complexes, wherein the gRNAs are complementary to targeted sites in the host nucleic acids, thereby generating host-nucleic acids adapter ligated only on one end and non-host nucleic acids adapter ligated on both the 5′ and 3′ ends; and amplifying the products using adapter-specific PCR or purifying, or treatment with exonuclease.
In another embodiment, provided herein is a method for serially depleting targeted nucleic acids in a sample comprising:(a) providing a sample comprising host nucleic acids and non-host nucleic acids, wherein the non-host nucleic acids comprise nucleic acids from at least one known non-host organism and nucleic acids from at least one unknown non-host organism; (b) contacting the sample with a plurality of CRISPR/Cas system protein-gRNA complexes, wherein the gRNAs are configured to hybridize to targeted sequences in the host nucleic acids, whereby a portion of the host nucleic acids are cleaved; (c) contacting the sample with a plurality of CRISPR/Cas system protein-gRNA complexes, wherein the gRNAs are configured to hybridize to targeted sequences in the at least one known non-host nucleic acids, whereby a portion of the at least one known non-host nucleic acids are cleaved; and (d) isolating the nucleic acids from the unknown non-host organism.
An exemplary application of the compositions and methods described herein is provided in
In various further processing steps occurring after the depletion targets have been cut or cleaved the mixture (1080) may be sized-selectively filtered to separate uncut fragments (1030) from cut fragments (1090) and (1095). Thereafter the uncut fragments (1030) can be amplified and sequenced. Alternately the amplification process can be used to sort the cut from the uncut segments since in some amplification systems only segments having an adapter (1020) at each end of the fragment is amplified.
Another exemplary application of the compositions and methods described herein is provided in
Depletion of Large Fragments of DNA Without Adapters
In some embodiments, the methods provided herein are used for depletion. However, instead of cleaving a library containing adapters, the guide RNAs are chosen to cleave multiple times in a pool of fragmented DNA of a large size (e.g., anywhere from 100 bp-10 kb). Following cleavage with CRISPR/Cas system protein-gRNA complexes, the DNA is subjected to size selection to remove small fragments (e.g., at least ½ or ⅓ of the average size of intact fragments). This is assisted, for example, by treatment with Lambda exonuclease, which is a 5′phosphate specific-exonuclease that proceeds in a 5′ to 3′ direction and would attack any fragments that had previously been cut with the CRISPR/Cas system protein. The resulting libraries can then be subjected to sequencing by single-molecule sequencers that do not require sequencing adapters; or adapters can be ligated for downstream analyses.
Depletion Using Thermostable CRISPR/Cas System Proteins
In another embodiment, a method of generating cleaved targeted sequences in a sample comprises: (a) providing a sample comprising sequences of interest and targeted sequences for cleavage; (b) contacting the sample with a plurality of CRISPR/Cas system protein-gRNA complexes, wherein the gRNAs are complementary to the targeted sequences, thereby generating cleaved targeted sequences; (c) dissociating the CRISPR/Cas system protein from the cleaved targeted sequences; (d) generating additional cleaved targeted sequences; and (c) recovering the uncut sequences of interest. In some embodiments, the CRISPR/Cas system protein is thermostable. In some embodiments, the dissociating of the CRISPR/Cas system protein from the cleaved targeted sequences is achieved by elevating the temperature of the mixture of step (b) to at least 75°. In some embodiments, the generating of additional cleaved targeted sequences is achieved by lowering the temperature of the mixture of step (b) to at least 50°.
In another embodiment, a method of depleting targeted sequences in a sample comprises: (a) providing a sample comprising sequences of interest and targeted sequences for depletion; (b) contacting the sample with a plurality of CRISPR/Cas system protein-gRNA complexes, wherein the gRNAs are complementary to the targeted sequences, thereby generating cleaved targeted sequences, and wherein the CRISPR/Cas system protein is thermostable; (c) elevating the temperature of the mixture of step (b) to at least 75°; (d) lowering the temperature of the mixture of step (b) at least 50°; repeating steps (c) and (d) at least once; and (f) recovering the uncut sequences of interest.
CRISPR/Cas System Protein-Mediated Depletion in the Presence of a CRISPR/Cas Protein Nickase
In some embodiments, depletion of unwanted, targeted nucleic acids can be carried out using a CRISPR/Cas system protein nickase. In one embodiment, the method comprises: making a DNA sequencing library comprising DNA to be removed (for example human DNA not of interest) and DNA of interest (for example an DNA from an unknown pathogen); designing guide RNAs so that all the DNA to be depleted will have two guide RNA binding sites in close proximity (for example, less than 15 bases apart) on opposite DNA strands; adding CRISPR/Cas system protein Nickase and guide RNAs to the DNA library. In this embodiment, the CRISPR/Cas system protein Nickase can recognize its target sites on the DNA to be removed and cuts only one strand. For DNA to be depleted, two separate CRISPR/Cas system protein Nickases can cut both strands of the DNA to be removed (e.g. human DNA) in close proximity; only the DNA to be removed (e.g. human DNA) will have two CRISPR/Cas system protein nickase sites in close proximity which creates a double stranded break. If a CRISPR/Cas system protein Nickase recognizes non-specifically or at low affinity a site on the DNA of interest (e.g. pathogen DNA), it can only cut one strand which would not prevent subsequent PCR amplification or downstream processing of the DNA molecule. This is pictorially depicted in
CRISPR/Cas System Protein-Mediated Depletion and Biotin Labeling
In some embodiments, the CRISPR/Cas system protein cleaved products are eliminated with the use of biotin. For example in a sample originally comprising <5% DNA of interest and >95% DNA targeted for depletion, the >95% DNA is fragmented and depleted, and the uncut (<5% DNA of interest) DNA comprises a biotin label is purified by binding to streptavidin beads. This example illustrates a method of removing unwanted DNA (depleted DNA fragmented) after CRISPR/Cas system protein-mediated cleavage without the use of an exonuclease.
An exemplary embodiment is depicted in
Exonuclease Treatment Following Depletion
In some embodiments, it is desirable to remove cut fragments prior to PCR amplification or sequencing.
In one embodiment, CRISPR/Cas system protein-mediated depletion is followed by an exonuclease treatment. The exonuclease treatment can further degrade a CRISPR/Cas system protein cleaved nucleic acid, while leaving the uncut nucleic acid comprising sequences of interest intact.
In one embodiment, a method of depleting targeted sequences in a sample comprises: providing a sample comprising sequences of interest and targeted sequences for depletion; contacting the sample with a plurality of CRISPR/Cas system protein-gRNA complexes, wherein the gRNAs are complementary to the targeted sequences, thereby generating cleaved targeted sequences; and contacting the product of step (b) with an exonuclease. In one embodiment the exonuclease is Exonuclease III. In another embodiment, the exonuclease is BAL-31.
In one exemplary embodiment, DNA is extracted from a biological sample and fragmented and a library, for example a sequencing library, comprising the fragments of DNA, is thus created. Y-shaped adapters or circular adapters are ligated to the DNA fragments. The DNA fragments comprise DNA sequences of interest, and targeted sequences (DNA not of interest). The library is contacted with a plurality of CRISPR/Cas system protein-gRNA complexes, and the CRISPR/Cas system protein cuts the target DNA not of interest and leaves the other DNA sequences of interest intact. The resulting products are contacted with Exonuclease III. Exonuclease III can initiate unidirectional 3′>5′ degradation of one DNA strand by using blunt end or 5′ overhangs, yielding single-stranded DNA and nucleotides; it is not active on single-stranded DNA, and thus 3′ overhangs, such as the Y-shaped adapter ends, are resistant to degradation. As a result, intact double-stranded DNA libraries uncut by the CRISPR/Cas system protein is not digested by Exonuclease III, while DNA molecules that have been cleaved by the CRISPR/Cas system protein are degraded by Exonuclease III with its 3′>5′ activity from the blunt ends cut by Cas9 towards the adaptors.
In another exemplary embodiment, Exonuclease BAL-31 is used degrade CRISPR/Cas system protein-cleaved DNA while leaving the uncut DNA of interest intact. In one exemplary embodiment, DNA is extracted from a biological sample and fragmented and a library, for example a sequencing library, comprising the fragments of DNA, is thus created. The DNA fragments comprise DNA sequences of interest, and targeted sequences (DNA not of interest). The 3′ ends of the fragments are tailed with poly-dG using terminal transferase. The library is contacted with a plurality of CRISPR/Cas system protein-gRNA complexes, and the CRISPR/Cas system protein cuts the target DNA not of interest and leaves the other DNA sequences of interest intact. The resulting products are contacted with Exonuclease BAL-31. Exonuclease BAL-31 has two activities: double-stranded DNA exonuclease activity, and single-stranded DNA/RNA endonuclease activity. The double-stranded DNA exonuclease activity allows BAL-31 to degrade DNA from open ends on both strands, thus reducing the size of double-stranded DNA. The longer the incubation, the greater the reduction in size of the double-stranded DNA, making it useful for depleting medium to large DNA (>200 bp). It was noted that the single-stranded endonuclease activity of BAL-31 allows it to digest poly-A, -C or -T very rapidly, but is extremely low in digesting poly-G (Marrone and Ballantyne, 2008). Because of this nature, adding single-stranded poly-dG at 3′ ends of the libraries serves as a protection from being degraded by BAL-31. As a result, DNA molecules that have been cleaved by a CRISPR/Cas system protein can be degraded by BAL-31 with its double-stranded DNA exonuclease activity from the double-stranded blunt end cut by the CRISPR/Cas system protein towards the other end carrying poly-dG, effecting depletion; while intact DNA libraries uncut by the CRISPR/Cas system protein are not digested by BAL-31 due to their 3′ end poly-dG protection.
Controls for Monitoring Depletion
In the embodiments discussed, it is desirable to provide positive and/or negative controls. A positive control can ensure depletion of target nucleic acids proceeds with fidelity.
In some embodiments, a control set of reagents is a positive control set of reagents, positive control target sequences. In an exemplary embodiment, the positive control set of reagents comprise a collection of nucleic acid fragments, wherein the fragments comprise the target sequences, to which the gRNAs are at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% complementary. This control could be run alongside a user's reaction to ensure that all components are working properly. After depletion with a CRISPR/Cas system protein, elimination of the positive control target sequences could be measured by gel electrophoresis or qPCR.
In some embodiments, the control set of reagents is a negative control set of reagents. In some embodiments a negative control ensures that off-target cutting is minimal or nonexistent. In an exemplary embodiment, the negative control set of reagents comprise a second set of gRNAs, wherein the second set of gRNAs exhibit reduced binding to the target sequences, as compared to the first set of gRNAs. In some embodiments, the second set of gRNAs exhibit 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 85% reduced binding to the target sequences, as compared to the first set of RNAs. In another exemplary embodiment, the negative control set of reagents comprise a second set of gRNAs, wherein the second set of gRNAs are no more than 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% complementary to target sequences. The negative control could be a set of gRNAs with less than 100% identity to the gRNAs used; for example, with 1, 2, 3, 4, 5, or more mismatches to the target-specific sequence. In some embodiments, the negative control set of reagents comprise a collection of nucleic acid fragments, wherein the fragments are no more than 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% complementary to the first set of gRNAs. The DNA library could have with less than 100% identity to the targeted sequences of gRNAs used in the kit; for example, with 1, 2, 3, 4, 5, or more mismatches to the complementary sequence of the gRNA. This control could be run alongside a user's reaction to ensure that all components are working properly and to measure any off-target activity of the enzyme. After depletion with CRISPR/Cas system protein, the amount of off-target depletion can be measured by gel electrophoresis or qPCR.
With respect to the positive and negative controls contemplated herein, complementarity of the gRNA to its target as provided herein can refer to the complementarity of the gRNA along the entire length of the target. In some embodiments, however the complementarity may be more impacted by mismatches of nucleic acids at the terminal end of the gRNA, at the area binding closest to the PAM sequence. Mismatches in this region may impact the capacity to bind the target in a greater manner than mismatches of nucleic acids elsewhere along the gRNA. Likewise, mismatches in the gRNA further away from the PAM sequence may impact the capacity to bind the target less than mismatches of nucleic acids elsewhere along the gRNA. In some embodiments, the negative control gRNA has a lower complimentarily near the PAM sequence, but higher complementarity farther away from the PAM sequence. For example, in some embodiments, the negative control gRNA has 50%-70% complementarity near the PAM sequence, but 70%-100% complementarity farther away from the PAM sequence. In some embodiments, the negative control gRNA has 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% complementarity along the entire length of the target.
Depleting Mitochondrial DNA Following ATAC-Seq
The Assay for Transposase-Accessible Chromatin Using Sequencing (ATAC-seq) technique is used in molecular biology to study chromatin, namely its accessible sites. The method is based on tagging open chromatin regions with sequencing adaptors by transposase, such that DNA fragments from these open chromatin regions can be amplified and subjected to high-throughput sequencing (WO2014/189957; Buenrostro et al., Nature Methods, Vol. 10, No. 12, December 2013, p. 1213). Although ATAC-seq requires much less starting material compared to other assays for genome-wide chromatin accessibility, one of the main drawbacks is that the sequencing library is often contaminated with high percentage of mitochondrial DNA (which is also open DNA), and requires many more sequencing reads for standard accessibility studies of the human genome, such as DNase-seq and FAIRE-seq (M. Tsompana and M J. Buck, 2014). Therefore there is a need in the art to remove unwanted mitochondrial DNA contamination from the sequencing library following an ATAC-Seq procedure, in order to decrease sequencing costs. The present disclosure provides a method to selectively deplete mitochondrial DNA from a sample that has undergone ATAC-Seq.
Provided herein is a method for analyzing genomic DNA, comprising: (a) treating DNA isolated from a population of cells with an insertional enzyme to produce a plurality of tagged fragments of non-mitochondrial genomic DNA, whereby also generating a residual amount of tagged mitochondrial DNA; (b) enriching the product of step (a) for non-mitochondrial DNA according to any of the enrichment or depletion methods provided herein. In some embodiments, the method further comprises sequencing at least some of the tagged fragments to produce a plurality of sequence reads; and making an epigenetic map of a region of the genome of said cells by mapping information obtained from the sequence reads to the region.
In another aspect, the present disclosure provides a method for assisting the determination of accessibility of a polynucleotide at a site, wherein the polynucleotide is from a cell sample, said method comprising: inserting a plurality of molecular tags with an insertional enzyme into the polynucleotide and using the molecular tags to determine accessibility at the site, and removing unwanted DNA contamination using the CRISPR/Cas system-based depletion methods described herein. The cell sample can be from a primary source. The cell sample may consist of a single cell. The cell sample may consist of a finite number of cells (e.g. less than about 500,000 cells).
The insertional enzyme can be any enzyme capable of inserting a nucleic acid sequence into a polynucleotide. In some cases, the insertional enzyme can insert the nucleic acid sequence into the polynucleotide in a substantially sequence-independent manner. The insertional enzyme can be prokaryotic or eukaryotic. Examples of insertional enzymes include, but are not limited to, transposases, HERMES, and HIV integrase. The transposase can be a Tn transposase (e.g. Tn3, Tn5, Tn7, Tn10, Tn552, Tn903), a MuA transposase, a Vibhar transposase (e.g. from Vibrio harveyi), Ac-Ds, Ascot-I, BsI, Cin4, Copia, En/Spm, F element, hobo, Hsmar1, Hsmar2, IN (HIV), ISI, IS2, IS3, IS4, IS5, IS6, ISIO, IS2I, IS30, IS50, ISSI, ISI50, IS256, IS407, IS427, IS630, IS903, IS911, IS982, IS103I, ISL2, LI, Mariner, P element, Tam3, Tel, Tc3, Tel, THE-I, Tn/O, TnA, Tn3, Tn5, Tn7, TnlO, Tn552, Tn903, Toll, Tol2, TnlO, Tyl, any prokaryotic transposase, or any transposase related to and/or derived from those listed above. In certain instances, a transposase related to and/or derived from a parent transposase can comprise a peptide fragment with at least about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% amino acid sequence homology to a corresponding peptide fragment of the parent transposase. The peptide fragment can be at least about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 60, about 70, about 80, about 90, about 100, about 150, about 200, about 250, about 300, about 400, or about 500 amino acids in length. For example, a transposase derived from Tn5 can comprise a peptide fragment that is 50 amino acids in length and about 80% homologous to a corresponding fragment in a parent Tn5 transposase. In some cases, the insertion can be facilitated and/or triggered by addition of one or more cations. The cations can be divalent cations such as, for example, Ca2+, Mg2+ and Mn2+.
The molecular tags can comprise sequencing adaptors, locked nucleic acids (LNAs), zip nucleic acids (ZNAs), RNAs, affinity reactive molecules (e.g. biotin, dig), self-complementary molecules, phosphorothioate modifications, azide or alkyne groups. In some cases, the sequencing adaptors can further comprise a barcode label. Further, the barcode labels can comprises a unique sequence. The unique sequences can be used to identify the individual insertion events. Any of the tags can further comprise fluorescence tags (e.g. fluorescein, rhodamine, Cy3, CyS, thiazole orange, etc.).
Kits and Articles of Manufacture
The present application provides kits comprising any one or more of the compositions described herein, not limited to adapters, gRNAs, gRNA libraries, and the like.
In one embodiment, the kit comprises a collection or library of gRNAs wherein the gRNAs are targeted to human DNA sequences. In another embodiment, the kit comprises a collection or library of gRNAs wherein the gRNAs are targeted to bovine DNA sequences. In another embodiment, the kit comprises a collection or library of gRNAs wherein the gRNAs are targeted to human ribosomal RNA sequences. In another embodiment, the kit comprises a collection or library of gRNAs wherein the gRNAs are targeted to human mitochondrial DNA sequences.
In some embodiments, the kit comprises: a CRISPR/Cas system protein and gRNAs, wherein the gRNAs are complementary to mitochondrial DNA; or wherein the gRNAs are complementary to the entire genome; or the wherein the gRNAs are complementary to a cDNA made from a whole transcriptome; or wherein the gRNAs are complementary to cDNA made form at least 10, at least 25, at least 50, at least 100, at least 250, at least 500, at least 750, at least 102, at least 103, at least 104, at least 105, at least 106, at least 107, at least 108, at least 109, at least 1010 unique transcripts; or wherein the gRNAs are complementary to cDNA made from the most abundant transcripts in a transcriptome, for example the most abundant 100, 75, 50, 25, 20, 15, or 10 transcripts in a transcriptome; or wherein the gRNAs are complementary to cDNA made from a subset of the most abundant 100, 75, 50, 25, 20, 15, or 10 transcripts in a transcriptome.
In some embodiments, the kit comprises: a CRISPR/Cas system protein and gRNAs, wherein the CRISPR/Cas system protein comprises a mixture of CRISPR/Cas system proteins from different bacteria; or wherein the CRISPR/Cas system protein is engineered; wherein the gRNAs are complementary to mitochondrial DNA; or wherein the gRNAs are complementary to genomic DNA, representing the entire genome; or wherein the gRNAs are complementary to cDNA libraries derived from the whole transcriptome; or wherein the gRNAs are complementary to cDNA libraries derived from at least 10, at least 25, at least 50, at least 100, at least 250, at least 500, at least 750, at least 102, at least 103, at least 104, at least 105, at least 106, at least 107, at least 108, at least 109, at least 1010 unique transcripts; or wherein the gRNAs are complementary to the cDNA libraries derived from the most abundant 100, 75, 50, 25, 20, 15, or 10 transcripts in a transcriptome; or wherein the gRNAs are complementary to the cDNA libraries derived from a subset of the most abundant 100, 75, 50, 25, 20, 15, or 10 transcripts in a transcriptome; or wherein the gRNAs comprise a mixture of gRNAs capable of targeting a CRISPR/Cas system protein from different bacterial species to the target sequences.
In some embodiments, the kit comprises: a CRISPR/Cas system protein; gRNAs, wherein the gRNAs are complementary to a target of interest; and an enzyme having exonuclease activity. In one embodiment, the exonuclease is Exonuclease III. In one embodiment, the exonuclease is BAL-31.
In some embodiments, the kit comprises: a CRISPR/Cas system protein, wherein the CRISPR/Cas system protein is thermostable; and gRNAs, wherein the gRNAs are complementary to a target of interest.
In some embodiments, the kit comprises: a CRISPR/Cas system protein; and a first set of gRNAs, wherein the gRNAs are complementary to target sequences of interest; and a control set of reagents. In one embodiment, the control set of reagents is a positive control set of reagents. In an exemplary embodiment, the positive control set of reagents comprise a collection of nucleic acid fragments, positive control target sequences, wherein the fragments comprise the target sequences, to which the gRNAs are complementary. In one embodiment, the control set of reagents is a negative control set of reagents. In an exemplary embodiment, the negative control set of reagents comprise a second set of gRNAs, wherein the second set of gRNAs exhibit reduced binding to the target sequences, as compared to the first set of gRNAs. In another exemplary embodiment, the negative control set of reagents comprise a collection of nucleic acid fragments, wherein the fragments are no more than 90% complementary to the first set of gRNAs.
In some embodiments, the kit comprises: reagents for isolating DNA from a population of cells; an insertional enzyme; a CRISPR/Cas system protein; and a plurality of gRNAs, wherein the gRNAs are complementary to mitochondrial DNA; or wherein the gRNAs are complementary to the entire genome; or the wherein the gRNAs are complementary to the whole transcriptome; or wherein the gRNAs are complementary to the top 100, 75, 50, 25, 20, 15, or 10 genes in a transcriptome.
The present application also provides articles of manufacture comprising any one of the kits described herein. Examples of an article of manufacture include vials (including sealed vials).
The following examples are included for illustrative purposes and are not intend to limit the scope of the invention.
Overview
As depicted in
Expression of Cas9
Cas9 (from S. pyogenes) was cloned into the pET30 expression vector (EMD biosciences) to insert the hexahistidine tag immediately upstream of the Cas9 start codon. The resulting plasmid was transformed into the Rosetta (DE3) BL21 bacterial strain (EMD biosciences) and grown in 1L of LB media with vigorous aeration until optical density of the culture (OD at 600 nm) reached 0.4. The temperature was lowered to 25° C., 0.2 mM IPTG was added and the culture grown for another four hours. Cells were then harvested by centrifugation (1,000×g for 20 min at 4° C.), resuspended in 10 ml binding buffer (20 mM Tris pH8, 0.5 M NaCl, 5 mM Imidazole, 0.05% NP40) and lysed by sonication (7×10 second bursts at 30% power, Sonifier 250, Branson). Insoluble cell debris were removed by centrifugation at 10,000×g for 20 min; supernatant containing soluble protein was then mixed with 0.4 ml of NTA beads (Qiagen) and loaded onto a column. Beads were washed three times with 4 ml binding buffer, then eluted with 3×0.5 ml of binding buffer supplemented with 250 mM Imidazole. Eluted fractions were then concentrated and buffer exchanged with storage buffer (10 mM Tris pH8, 0.3 M NaCl, 0.1 mM EDTA, 1 mM DTT, 50% glycerol) using a 30,000 MWCO protein concentrator (Life Technologies), verified by SDS PAGE followed by Colloidal Blue staining (Life Technologies), quantified, then stored at −20° C. for later use.
A mutant Cas9 nickase, a D10A mutant of S. pyogenes Cas9, was produced and purified using the same procedures used to produce Cas9 as above.
Preparation of Guide RNA Libraries
Twenty-five constructs were designed with each one containing the T7 RNA polymerase promoter, 20 base pairs of mitochondrial DNA specific sequence, and the guide RNA scaffold from S. pyogenes. For each construct two oligonucleotides were ordered (see Tables 1 and 2 for sequences) and combined at 1 μM final concentration in 5 μl, then heated at 98° C. for 3 minutes, then cooled at a rate of 5° C./minute to 55° C., and finally incubated at 55° C. for 5 minutes.
Annealed oligos were then ligated to the T7GRNE vector which contains the guide RNA scaffold by golden gate cloning; annealed oligos were combined to 500 ng of T7GRNE plasmid, 1 μl of BsaI restriction enzyme and 1 μl of T7 DNA ligase in a total volume of 20 μl. Reactions were incubated in a thermal cycler for 10 cycles of 37° C. for 15 minutes followed by 20° C. for 10 minutes, followed by a single cycle at 80° C. for 15 minutes. Products were then PCR amplified using the specific forward primer (see Tables 1 and 2) and the reverse primer (5′-CAGAGCGAGGTATGTAGG-3′). Cycling conditions were as follows: 30 cycles of 95° C. for 1 min, 57° C. for 30 s, and 72° C. for 90 s. Successful reactions were confirmed by the presence of a 1.6 kb fragment by agarose gel electrophoresis and SYBR safe staining. Successful reactions were pooled together and purified using a PCR cleanup kit (Thermo Fisher Scientific).
Guide RNAs were designed using the following rules:
Rule 1: Found the following sequence (5′>3′ direction) in the genomic region of interest (for example the entire exome or mitochondrial genome) on any strand:
NNNNNNNNNNNNNNNNNNNNNGG
Rule 2: Underlined sequence (20-mer) is the targeting part of guide RNA included in the oligonucleotide ordered.
GCCTCGAGCTAATACGACTCACTATAG(G)
NNNNNNNNNNNNNNNNNNNN
To transcribe the guide libraries into guide RNA, we assembled the following in vitro transcription reaction mixture: 10 μl purified library (˜500 ng), 6.5 μl of H2O, of 2.25 μl of ATP, 2.25 μl of CTP, 2.25 μl of GTP, 2.25 μl of UTP, 2.25 μl 10× reaction buffer (NEB) and 2.25 μl of T7 RNA polymerase mix. The reaction was incubated at 37° C. for 24 hours, then diluted 10-fold in H2O. A single reaction produced ˜40 μg of RNA. The yield and size of the RNA (˜150 base pairs) was checked by running 1 μl of the reaction on a 5% TBE/Urea gel and staining with SYBR Gold (Life Technologies).
DNA-Specific Cas9-Mediated Depletion
Diluted guide RNA (1 μl, equivalent to 2 pmol) was combined with 3 μl 10× Cas9 reaction buffer (NEB), 20 μl H2O and 1 μl of recombinant Cas9 enzyme (NEB, 1 pmol/μl). A control reaction using a control guide RNA targeting the following sequence (5′-GGATTTATACAGCACTTTAA-3′) was performed separately, using the same parameters. This sequence is absent from either the human chromosomal or mitochondrial DNA. Reactions were incubated for 15 min at 37° C., then supplemented with 5 μl diluted DNA library (50 pg/μl) and incubation at 37° C. continued for 90 min. The reactions were terminated by adding RNase A (Thermo Fisher Scientific) at a 1:100 dilution, heating to 98° C. for 5 min, then cooling to room temperature and adding 100 μl H2O. Reactions were then stored at −20° C. until use.
Quantification of Mitochondrial DNA after Depletion
For each reaction (each test condition and the reaction using the control guide RNA) two separate 5 μl samples were analyzed by real time PCR using both control and test primers. For control primers reactions, samples were incubated with 2 μl H2O, 0.25 μl of 10 μM primer P5 (5′-AATGATACGGCGACCACCGA-3′), and 0.25 μl of 10 μM P7, (5′-CAAGCAGAAGACGGCATACGA-3′) and 7.5 μl of 2× Maxima SYBR Green master mix (Thermo Fisher Scientific). For test primers reactions, samples were incubated with 1.75 μl H2O, 0.5 μl of 10 μM mitochondrial primers (see tables 1 and 2), and 0.25 μl of 10 μM P7, (5′-CAAGCAGAAGACGGCATACGA-3′) and 7.5 μl of 2× Maxima SYBR Green master mix (Thermo Fisher Scientific). Reactions were incubated in an iCycler real-time PCR thermal cycler (BioRad) using the following 2-step cycling conditions: 95° C. for 3 min, followed by 40 cycles of 95° C. for 10 s, 55° C. for 45 s.
For the two real time PCR conditions, using control and test primers, a calibration curve was performed. The diluted DNA library (50 μg/μl) was further diluted 1:10, 1:100,1:1000, 1:10,000 and these dilutions were analyzed by real time PCR using the same reaction conditions, instrument and cycling conditions described above.
The amount of total DNA and mitochondrial DNA was deduced from the results of the control and test primers reactions, respectively. For each DNA library depletion experiment, the ratio of mitochondrial DNA:total DNA was determined and normalized to the experiment using the control guide RNA, then plotted as a column graph shown in
TABLE 2
Forward primers-used in both the annealing
and subsequent PCR reactions
Oligo Name
Sequence
T7-13-F
GCC TCG AGC TAA TAC GAC TCA CTA TAG
GCT CTT AAA ACT AGG CGG CTA G
T7-39-F
GCC TCG AGC TAA TAC GAC TCA CTA TAG
ATT TAC ACT CAC AAC ACC CTG
T7-41-F
GCC TCG AGC TAA TAC GAC TCA CTA TAG
AAC AGC TAT CCA TTG GTC TTG
T7-43-F
GCC TCG AGC TAA TAC GAC TCA CTA TAG
GCA GCC GGA AGC CTA TTC GCG
T7-61-F
GCC TCG AGC TAA TAC GAC TCA CTA TAG
GTA ATG AGG ATG TAA GCC CGG
T7-63-F
GCC TCG AGC TAA TAC GAC TCA CTA TAG
ATA TTT ACA AGA GGA AAA CCG
T7-65-F
GCC TCG AGC TAA TAC GAC TCA CTA TAG
GTT TGA AGC TTA GGG AGA GCT G
T7-67-F
GCC TCG AGC TAA TAC GAC TCA CTA TAG
GTA TGG CTT TGA AGA AGG CGG
T7mtgRNA3
GCC TCG AGC TAA TAC GAC TCA CTA TAG
TAG ATG ACG GGT TGG GCC AGG
T7mtgRNA7
GCC TCG AGC TAA TAC GAC TCA CTA TAG
AGC TTT ACA GTG GGC TCT AGG
T7mtgRNA11
GCC TCG AGC TAA TAC GAC TCA CTA TAG
ATG GCA GCT TCT GTG GAA CGG
T7mtgRNA15
GCC TCG AGC TAA TAC GAC TCA CTA TAG
GTG GTA AGG GCG ATG AGT GTG
T7mtgRNA31
GCC TCG AGC TAA TAC GAC TCA CTA TAG
TCC ATA ACG CTC CTC ATA CTG
T7mtgRNA33
GCC TCG AGC TAA TAC GAC TCA CTA TAG
TCT CCC TTC ACC ATT TCC GAG
T7mtgRNA35
GCC TCG AGC TAA TAC GAC TCA CTA TAG
GCC TAT GAG TGA CTA CAA AAG
T7mtgRNA37
GCC TCG AGC TAA TAC GAC TCA CTA TAG
CTT TGC CGC CTG CGA AGC AGG
T7mtgRNA57
GCC TCG AGC TAA TAC GAC TCA CTA TAG
ATG TCT CCA TCT ATT GAT GAG
T7mtgRNA79
GCC TCG AGC TAA TAC GAC TCA CTA TAG
GGA GGC CTG CCC CCG CTA ACG
T7mtgRNA81
GCC TCG AGC TAA TAC GAC TCA CTA TAG
CGA GCC GAG CTG GGC CAG CCG
T7mtgRNA83
GCC TCG AGC TAA TAC GAC TCA CTA TAG
GCG AGC AGG AGT AGG AGA GAG
T7mtgRNA85
GCC TCG AGC TAA TAC GAC TCA CTA TAG
CAA CAC TTT CTC GGC CTA TCG
T7mtgRNA87
GCC TCG AGC TAA TAC GAC TCA CTA TAG
ACT TTG ACA AAG TTA TGA AAG
T7mtgRNA89
GCC TCG AGC TAA TAC GAC TCA CTA TAG
TCA AAT CAA TTG GCC ACC AAG
T7mtgRNA91
GCC TCG AGC TAA TAC GAC TCA CTA TAG
ACT CAT TCA ACC AAT AGC CCG
T7mtgRNA93
GCC TCG AGC TAA TAC GAC TCA CTA TAG
GTG CTC ACA CGA TAA ACC CTG
TABLE 2
Reverse primers-used during the anneal
reaction and the real time PCR reactions
Oligo Name
Sequence
mtgRNA2
aaaacTTCTAAACGCTAATCCAAGCct
mtgRNA14
aaaacTAGCCGCCTAGTTTTAAGAGct
mtgRNA40
aaaacAGGGTGTTGTGAGTGTAAATct
mtgRNA42
aaaacAAGACCAATGGATAGCTGTTct
mtgRNA44
aaaacGCGAATAGGCTTCCGGCTGCct
mtgRNA62
aaaacCGGGCTTACATCCTCATTACct
mtgRNA64
aaaacGGTTTTCCTCTTGTAAATATct
mtgRNA66
aaaacAGCTCTCCCTAAGCTTCAAAct
mtgRNA68
aaaacCGCCTTCTTCAAAGCCATACct
mtgRNA4
aaaacCTGGCCCAACCCGTCATCTAct
mtgRNA8
aaaacCTAGAGCCCACTGTAAAGCTct
mtgRNA12
aaaacCGTTCCACAGAAGCTGCCATct
mtgRNA16
aaaacACACTCATCGCCCTTACCACct
mtgRNA32
aaaacAGTATGAGGAGCGTTATGGAct
mtgRNA34
aaaacTCGGAAATGGTGAAGGGAGAct
mtgRNA36
aaaacTTTTGTAGTCACTCATAGGCct
mtgRNA38
aaaacCTGCTTCGCAGGCGGCAAAGct
mtgRNA58
aaaacTCATCAATAGATGGAGACATct
mtgRNA80
aaaacGTTAGCGGGGGCAGGCCTCCt
mtgRNA82
aaaacGGCTGGCCCAGCTCGGCTCGct
mtgRNA84
aaaacTCTCTCCTACTCCTGCTCGCct
mtgRNA86
aaaacGATAGGCCGAGAAAGTGTTGct
mtgRNA88
aaaacTTTCATAACTTTGTCAAAGTct
mtgRNA90
aaaacTTGGTGGCCAATTGATTTGAct
mtgRNA92
aaaacGGGCTATTGGTTGAATGAGTct
mtgRNA94
aaaacAGGGTTTATCGTGTGAGCACct
Overview
As depicted in
The pre- and post-capture libraries were quantified by real time PCR using two different sets of primers: P5 with P7, which amplifies all DNA library fragments or mitochondrial DNA specific primers which only amplify mitochondrial DNA. The ratio of these two separate reactions was used to quantify the depletion of mitochondrial DNA
The pre- and post-capture libraries were also sequenced on an Illumina MiSeq platform, and the resulting reads were mapped to the human genome to determine mitochondrial DNA content.
Expression of Cas9
Cas9 (from S. pyogenes) was cloned into the pET30 expression vector (EMD biosciences) to insert the hexahistidine tag immediately upstream of the Cas9 start codon. The resulting plasmid was transformed into the Rosetta (DE3) BL21 bacterial strain (EMD biosciences) and grown in 1L of LB media with vigorous aeration until optical density of the culture (OD at 600 nm) reached 0.4. The temperature was lowered to 25° C., 0.2 mM IPTG was added and the culture grown for another four hours. Cells were then harvested by centrifugation (1,000×g for 20 min at 4° C.), resuspended in 10 ml binding buffer (20 mM Tris pH8, 0.5 M NaCl, 5 mM Imidazole, 0.05% NP40) and lysed by sonication (7×10 second bursts at 30% power, Sonifier 250, Branson). Insoluble cell debris were removed by centrifugation at 10,000×g for 20 min; supernatant containing soluble protein was then mixed with 0.4 ml of NTA beads (Qiagen) and loaded onto a column. Beads were washed three times with 4 ml binding buffer, then eluted with 3×0.5 ml of binding buffer supplemented with 250 mM Imidazole. Eluted fractions were then concentrated and buffer exchanged with storage buffer (10 mM Tris pH8, 0.3 M NaCl, 0.1 mM EDTA, 1 mM DTT, 50% glycerol) using a 30,000 MWCO protein concentrator (Life Technologies), verified by SDS PAGE followed by Colloidal Blue staining (Life Technologies), quantified, then stored at −20° C. for later use.
A mutant Cas9 nickase, a D10A mutant of S. pyogenes Cas9, was produced and purified using the same procedures used to produce Cas9 as above.
Preparation of Guide RNA Libraries
T7-guideRNA oligonucleotides (Table 3) and a separate oligonucleotide, stlgR (sequence, GT TTT AGA GCT AGA AAT AGC AAG TTA AAA TAA GGC TAG TCC GTT ATC AAC TTG AAA AAG TGG CAC CGA GTC GGT GCT TTT TTT GGA TCC GAT GC) were ordered and synthesized (IDT).
The stlgR oligonucleotide (300 pmol) was sequentially 5′ phosphorylated using T4 PNK (New England Biolabs) and then 5′ adenylated sing the 5′adenylation kit (New England Biolabs), according to the manufacturer's instructions. T7-guide RNAs oligonucleotides (5 pmol) and the 5′adenylated stlgR (10 pmol) were then ligated using thermostable 5′App DNA/RNA ligase (New England Biolabs) at 65C for one hour. Ligation reactions were heat inactivated at 90° C. for 5 min, then amplified by PCR (using OneTaq, New England Biolabs, 30 cycles of 95° C. 30 secs, 57° C. 20 secs, 72° C., 20 secs) with primers ForT7 (sequence GCC TCG AGC TAA TAC GAC TCA C) and gRU (sequence AAAAAAAGCACCGACTCGGTG). PCR products were purified using PCR cleanup kit (Life Technologies) and verified by agarose gel electrophoresis and sequencing.
Verified products were then used as templates for in vitro transcription reactions using the HiScribe T7 transcription kit (New England Biolabs). 500-1000 ng of template was incubated overnight at 37° C. according to the manufacturer's instruction. The resulting guide RNA(s) was purified using the RNA cleanup kit (Life Technologies), eluted into 100 μl of RNase-free water, quantified and stored at −20° C. until use.
In Vitro Transcription
To transcribe the guide libraries into guide RNA, we assembled the following in vitro transcription reaction mixture: 10 μl purified library (˜500 ng), 6.5 μl of H2O, of 2.25 μl of ATP, 2.25 μl of CTP, 2.25 μl of GTP, 2.25 μl of UTP, 2.25 μl 10× reaction buffer (NEB) and 2.25 μl of T7 RNA polymerase mix. The reaction was incubated at 37° C. for 24 hours, then diluted 10-fold in H2O. A single reaction produced ˜40 μg of RNA. The yield and size of the RNA (˜150 base pairs) was checked by running 1 μl of the reaction on a 5% TBE/Urea gel and staining with SYBR Gold (Life Technologies).
DNA-Specific Cas9-Mediated Depletion
Multiple guide RNAs (from single or multiple in vitro transcription reactions) were selected to ensure that >95% of target DNA to be depleted contained at least one guide RNA target sequence. This can be computed by measuring the distribution of library size and determining the mean, N and the standard deviation SD; N-2SD=minimum size for >95% of the library, ensuring that there is one guide RNA per this size to ensure >95% depletion. This can also be described as the Maximum distance between guide RNAs=Mean of library size−2×(standard deviation of library size). In the case of the mitochondrial library, the library contains DNA fragments that are 450 base pairs in average length, with a standard deviation of 100. This results in 250 bp between guide RNAs at a maximum. Here, the DNA fragments in these libraries are 450 bp in average length (with an estimated standard deviation of 100 bp), <5% of DNA is smaller than 250 bp assuming a normal distribution. Assuming guide RNAs will target 100% of their assigned targets, >95% of target DNA should contain at least one guide RNA site and be depleted.
In this example, all guides constructed in example 2 in addition to those constructed in example 1 were used. These multiple guide RNAs were mixed in equimolar amounts to add to 80 pmol total, then combined with 40 pmol purified Cas9 protein in 60 μl Cas9 reaction buffer (20 mM HEPES pH 6.5, 100 mM NaCl, 5 mM MgC12, 0.1 mM EDTA) and incubated for 10 min at 37° C. 5 ng of DNA mixture (containing the desired DNA and the target DNA to be depleted) was added and the incubation continued for another 20 min. The reaction was stopped by adding 140 μl of TE buffer (10 mM Tris pH 8, 1 mM EDTA) containing 2 μg/ml RNase A and incubating for 20 min at 37° C. DNA was recovered using the PCR cleanup kit (Life Technologies) without any addition of isopropanol prior to column binding, and eluted in 20 μl of 10 mM Tris pH 8. Eluted DNA was re-amplified using the HiFidelity master mix (New England Biolabs) according to the manufacturer's instructions, with the appropriate primers (for example P5 and P7 for re-amplifying Illumina based DNA sequencing libraries) and for 8-13 cycles of PCR.
Sequencing and Bioinformatics
PCR products were purified using the PCR cleanup kit and used as templates for high-throughput DNA sequencing (Illumina MiSeq or NextSeq).
Fastq files were aligned to the human genome reference (hg20) using bwa or bowtie to determine proportions of reads mapping to the nuclear vs. mitochondrial genomes.
TABLE 3
Additional guide RNAs used in Example 2
Oligo Name
Sequence
mtgRNA97
GCC TCG AGC TAA TAC GAC TCA CTA TAG
GTC AGT GGG GGT GAG GTA AAA
mtgRNA99
GCC TCG AGC TAA TAC GAC TCA CTA TAG
GCT GAT CCT CCA AAT CAC CAC
mtgRNA101
GCC TCG AGC TAA TAC GAC TCA CTA TAG
GTT GGT ATC CTA GTG GGT GAG
mtgRNA103
GCC TCG AGC TAA TAC GAC TCA CTA TAG
GTT AGC CTA GCC ACA CCC CCA
mtgRNA105
GCC TCG AGC TAA TAC GAC TCA CTA TAG
GGC CGT TAA ACA TGT GTC AC
mtgRNA107
GCC TCG AGC TAA TAC GAC TCA CTA TAG
GTT AGC GAT GGA GGT AGG AT
mtgRNA109
GCC TCG AGC TAA TAC GAC TCA CTA TAG
GTA TAA CCT CCC CCA AAA TTC
mtgRNA111
GCC TCG AGC TAA TAC GAC TCA CTA TAG
GCA TTG CGA GAC GCT GGA GC
mtgRNA113
GCC TCG AGC TAA TAC GAC TCA CTA TAG
AAA AGA TAA AAT TTG AAA TC
mtgRNA115
GCC TCG AGC TAA TAC GAC TCA CTA TAG
GGT TTA TCG ATT ACA GAA C
mtgRNA117
GCC TCG AGC TAA TAC GAC TCA CTA TAG
AAA TCT CCT AAG TGT AAG TT
mtgRNA119
GCC TCG AGC TAA TAC GAC TCA CTA TAG
GGG ATT TAG AGG GTT CTG T
mtgRNA121
GCC TCG AGC TAA TAC GAC TCA CTA TAG
GAA AGG TTA AAA AAA GTA AA
mtgRNA123
GCC TCG AGC TAA TAC GAC TCA CTA TAG
AGG GTT TAC GAC CTC GAT GT
mtgRNA125
GCC TCG AGC TAA TAC GAC TCA CTA TAG
GAG GGT GAT GGT AGA TGT GG
mtgRNA127
GCC TCG AGC TAA TAC GAC TCA CTA TAG
GAT AAA TCA TAT TAT GGC CA
mtgRNA129
GCC TCG AGC TAA TAC GAC TCA CTA TAG
GGA ATG CTG GAG ATT GTA A
mtgRNA131
GCC TCG AGC TAA TAC GAC TCA CTA TAG
AAA TTA AGA ATG GTT ATG TT
mtgRNA133
GCC TCG AGC TAA TAC GAC TCA CTA TAG
GTT TGT GGG TTT AAG TCC CAT
mtgRNA135
GCC TCG AGC TAA TAC GAC TCA CTA TAG
GTG ATG CCA GCA GCT AGG AC
mtgRNA137
GCC TCG AGC TAA TAC GAC TCA CTA TAG
GTT CAC CGTAGG TGG CCT GAC
mtgRNA139
GCC TCG AGC TAA TAC GAC TCA CTA TAG
TGG AGG GTT CTT CTA CTA TT
mtgRNA141
GCC TCG AGC TAA TAC GAC TCA CTA TAG
GTG TTA GGA AAA GGG CAT AC
mtgRNA143
GCC TCG AGC TAA TAC GAC TCA CTA TAG
ATC TGT TTT TAA GCC TAA TG
mtgRNA145
GCC TCG AGC TAA TAC GAC TCA CTA TAG
GTT GTG TTT AAT ATT TTT AGT
mtgRNA147
GCC TCG AGC TAA TAC GAC TCA CTA TAG
GTG GTT GGT GTA AAT GAG TG
mtgRNA149
GCC TCG AGC TAA TAC GAC TCA CTA TAG
GAA AAC GTA GGC TTG GAT TA
mtgRNA151a
GCC TCG AGC TAA TAC GAC TCA CTA TAG
GGC CAG TGC CCT CCT AAT TG
mtgRNA153a
GCC TCG AGC TAA TAC GAC TCA CTA TAG
AAA TCC ACC CCT TAC GAG TG
mtgRNA155a
GCC TCG AGC TAA TAC GAC TCA CTA TAG
GCA TTT ACC ATC TCA CTT CT
mtgRNA157a
GCC TCG AGC TAA TAC GAC TCA CTA TAG
ATA AAA TAT GAT TAG TTC TG
mtgRNA159a
GCC TCG AGC TAA TAC GAC TCA CTA TAG
GTA GGA GAG TGA TAT TTG ATC
mtgRNA161a
GCC TCG AGC TAA TAC GAC TCA CTA TAG
GGT ATG AGT TTT TTT TGT TA
mtgRNA163 a
GCC TCG AGC TAA TAC GAC TCA CTA TAG
CCG ATG AAC AGT
mtgRNA165 a
GCC TCG AGC TAA TAC GAC TCA CTA TAG
AGT GTT AGA GTT TGG ATT AG
mtgRNA167a
GCC TCG AGC TAA TAC GAC TCA CTA TAG
GTT TGT GTA TGA TAT GTT TG
mtgRNA169a
GCC TCG AGC TAA TAC GAC TCA CTA TAG
GGA GTA GGG TTA GGA TGA G
mtgRNA171a
GCC TCG AGC TAA TAC GAC TCA CTA TAG
GAC CTA GTT CAA TGA ATC TG
mtgRNA173a
GCC TCG AGC TAA TAC GAC TCA CTA TAG
GGA CGC CTC CTA GTT TGT TA
mtgRNA175a
GCC TCG AGC TAA TAC GAC TCA CTA TAG
GCA GGT GTC TCC TCT ATC TT
mtgRNA177a
GCC TCG AGC TAA TAC GAC TCA CTA TAG
GAT TCA AAT TAT GTG TTT TT
mtgRNA179a
GCC TCG AGC TAA TAC GAC TCA CTA TAG
TCA TCT CCG GTT TAC AAG AC
Transcriptome-Wide gRNA Library:
Guide RNAs are generated, which are tiled across the entire transcriptome. The design is based on the blood transcriptome. A pipeline for the computational selection of guide RNAs spaced every ˜100-200 bp across each transcript is developed, based on the knowledge that there is expected to be an NGG site every ˜10-20 bp in the human genome on average). In this way, given a typical RNA-seq library insert size of ˜200-500 bp, each insert is expected to contain a target site. Following completion of the design, oligo pools containing the guide RNA target sequences and surrounding motifs are ordered, allowing for subsequent addition of a T7 promoter and the invariant portion of the guide RNA sequence. This approach enables the regeneration of the libraries for a period of time before needing to order new pools. Based on an array size of 90,000 elements, it is expected that guide RNAs spaced every ˜200 bp across the transcriptome with two arrays (assuming a transcriptome size of ˜30M bases) will be generated. These guide RNAs are then used along with Cas9 to deplete all human DNA from an RNA-seq library as described in the previous examples. Depletion and sequencing data analysis is done as described for the previous examples.
Validation:
The described approach is tested on test RNA-seq libraries. These RNA-seq libraries are made from a mixture of human and E. coli RNA in order to simulate the types of sequences being targeted. Following depletion of the library, sequencing is performed, to get ˜10× coverage of the transcriptome. As above, the proportion of all sequences mapping to human before and after depletion is measured; and the Kruskal-Wallis one-way ANOVA test can be used to determine statistical significance of depletion.
It is expected that this method, with the optimal gRNA designs, adjusting levels of Cas9 in the reaction, will achieve 90% depletion of non-ribosomal human sequences from an RNA-seq library, with less than 5% of non-human sequences being depleted.
A human blood sample carrying a known pathogen (e.g., Ebola) or an unknown pathogen is obtained and RNA is extracted (e.g., using a PAXgene Blood RNA extraction kit, Qiagen) and converted into a sequencing library using standard methods (e.g., using a KAPA RNA-seq library preparation kit). After cDNA creation and adapter ligation, the library is then mixed with Cas9 and a guide RNA pool targeting every ˜200 bases in the human transcriptome. The reaction is incubated at 37C for 20 minutes, allowing any fragments containing the target sequences to be cleaved by Cas9. The library is then purified and amplified using adapter-specific primers. Human sequences are effectively depleted. Only uncleaved sequences are amplified, and the resulting library is enriched for viral or pathogen-derived sequences.
In this example, the method is used to monitor viruses and other pathogens in milk and livestock.
RNA from cow milk or bovine (cow) blood is extracted and converted into a sequencing library as described in the above example. After cDNA creating and adapter ligation, the library is mixed with Cas9 and a guide RNA pool targeting ˜200 bases in the Bos taurus transcriptome. The reaction is incubated at 37C for 20 minutes, allowing any fragments containing the target sequences to be cleaved by Cas9. The library is then purified and amplified using adapter-specific primers. Bovine sequences are effectively depleted. Only uncleaved sequences are amplified, and the resulting library is enriched for viral or pathogen-derived sequences. In this scenario, the cow-depleted sequencing library can be sequenced and used to monitor viruses and other pathogens in milk and livestock.
This example shows a method that supports depletion without the need for adapters. In this example, the method is used for depletion experiments as described in the examples above. However, instead of cleaving a library containing adapters, the guide RNAs are chosen to cleave multiple times in a pool of fragmented DNA of large size (e.g., anywhere from 100 bp-10 kb). Following cleavage with Cas9/gRNA complexes, the DNA is subjected to size selection to remove small fragments (e.g., at least ½ or ⅓ of the average size of intact fragments). This could be assisted, for example, by treatment with Lambda exonuclease, which is a 5′phosphate specific-exonuclease that proceeds in a 5′ to 3′ direction and would attack any fragments that had previously been cut with Cas9. The resulting libraries can then be subjected to sequencing single-molecule sequencers that do not require sequencing adapters; or adapters can be ligated for downstream analyses.
In some embodiments, it is desirable to remove cut fragments prior to PCR amplification or sequencing. This example illustrates a method of removing unwanted DNA after Cas9-mediated cleavage, by using Exonuclease III to degrade cleaved DNA while leaving the uncut DNA of interest intact.
As depicted in
Exonuclease III can initiate unidirectional 3′>5′ degradation of one DNA strand by using blunt end or 5′ overhangs, yielding single-stranded DNA and nucleotides; it is not active on single-stranded DNA, and thus 3′ overhangs, such as the Y-shaped adapter ends, are resistant to degradation. As a result, intact double-stranded DNA libraries uncut by Cas9 (for example in this case, non-mitochondrial DNA) is not be digested by Exonuclease III, while DNA molecules that have been cleaved by Cas9 (for example in this case, mitochondrial DNA) is degraded by Exonuclease III with its 3′>5′ activity from the blunt ends cut by Cas9 towards the adaptors.
Thus unwanted DNA is digested. The remaining intact double-stranded DNA libraries are then recovered using the GeneJET PCR Purification Kit (Thermo Fisher Scientific) and eluted in 20 μl10 mM Tris-Cl pH 8. Samples are then quantified using Qubit Fluorometer (Life Technologies) and sequenced on the MiSeq system (Illumina).
Alternatively, unwanted DNA can be digested, as above, while intact DNA is recovered, for example, by column purification or PCR-amplification.
In some embodiments, it is desirable to remove cut fragments prior to PCR amplification or sequencing. This example illustrates an alternative method of removing unwanted DNA after Cas9-mediated cleavage, by using Exonuclease Bal-31 to degrade cleaved DNA while leaving the uncut DNA of interest intact.
As depicted in
Exonuclease Bal-31 has two activities: double-stranded DNA exonuclease activity, and single-stranded DNA/RNA endonuclease activity. The double-stranded DNA exonuclease activity allows BAL-31 to degrade DNA from open ends on both strands, thus reducing the size of double-stranded DNA. The longer the incubation, the greater the reduction in size of the double-stranded DNA, making it useful for depleting medium to large DNA (>200 bp). It is noted that the single-stranded endonuclease activity of BAL-31 allows it to digest poly-A, -C or -T very rapidly, but is extremely low in digesting poly-G (Marrone and Ballantyne, 2008). Because of this nature, adding single-stranded poly-dG at 3′ ends of the libraries can serve as a protection from being degraded by BAL-31, which we have verified using PCR products. As a result, DNA molecules that have been cleaved by Cas9 (for example in this case, mitochondrial DNA) will be degraded by BAL-31 with its double-stranded DNA exonuclease activity from the double-stranded blunt end cut by Cas9 towards the other end carrying poly-dG, effecting depletion; while intact DNA libraries uncut by Cas9 (for example in this case, non-mitochondrial DNA) will not be digested by BAL-31 due to their 3′ end poly-dG protection.
After Exonuclease Bal-31 incubation, the reaction mixture is heat inactivated at 75° C. for 20 min, and DNA is recovered using the GeneJET PCR Purification Kit (Thermo Fisher Scientific). Samples are then quantified using Qubit Fluorometer (Life Technologies) and sequenced on the MiSeq system (Illumina).
Alternatively, unwanted DNA can be digested, as above, while intact DNA is recovered, for example, by column purification.
In some embodiments, the Cas9 cleaved products are eliminated (for example the >95% mitochondrial DNA is eliminated) and the uncut (<5% DNA of interest) DNA comprises a biotin label is purified by binding to streptavidin beads. This example illustrates a method of removing unwanted DNA after Cas9-mediated cleavage without using an exonuclease, and is depicted in
A DNA mixture (containing >95% unwanted human DNA, and <5% other DNA of interest) was fragmented, end-repaired and ligated to adapters following regular protocols, then amplified by PCR using primers of Biotin-P5 (5′Biotin-AATGATACGGCGACCACCGA) and P7 (5′-CAAGCAGAAGACGGCATACGA). This ensures that the entire sequencing library possesses a 5′Biotin label only on one end of the DNA molecules. The DNA mixture is then subjected to Cas9 digestion, complexed with, for example for this case, a guide RNA library against mitochondrial DNA (the unwanted DNA), as described earlier. The Cas9 reaction is diluted 1:3 with TE (10 mM Tris-Cl pH 8, 1 mM EDTA) supplemented with 0.5% RNase A (Thermo Fisher Scientific), and incubated for 5 min at 37° C. Proteinase K (NEB) is then added at a final concentration of 8 units/ml and incubated for 5 min at 37° C. DNA molecules that are cleaved by Cas9 (for example in this case, mitochondrial DNA) will lose the Biotin labeled adaptor, thus cannot be sequenced or PCR amplified; while intact DNA libraries uncut by Cas9 (for example in this case, non-mitochondrial DNA of interest) will still carry the Biotin label. As a result, intact DNA uncut by Cas9 can then be recovered by adding streptavidin beads (Dynabeads MyOne Streptavidin C1, Thermo Fisher Scientific) to a final concentration of 2%. Beads are captured using a magnetic stand and washed 4 times with TE buffer. DNA is released from the beads by heating to 95° C. for 3 min, followed by 6 cycles of PCR using P5 and P7 primers (non-biotinylated). DNA is then purified using the GeneJET PCR Purification Kit (Thermo Fisher Scientific). Samples are then quantified using Qubit Fluorometer (Life Technologies) and sequenced on the MiSeq system (Illumina).
Although Cas9 can be used in combination with a library of guide RNAs to efficiently deplete a collection of target DNA, large amounts (>30 pmoles) of Cas9 and guide RNAs are needed. The reason for this usually >100 fold excess amount over target DNA is that, unlike classical restriction enzymes such as EcoRI, which detach completely from their target DNA after cleavage, Cas9 is not recycled after completion of the cutting reaction. Cas9 can remain tightly bound to one of the two daughter DNA product molecules (see
Two exemplary methods to make Thermostable Cas9 are provided.
The first method is isolation of thermostable Cas9. The gene for thermostable Cas9 and its corresponding guide RNA will be identified by sequence homology in the genome of thermophilic bacteria Streptococcus thermophilus and Pyrococcus furiosus. Cas9 genes will then be cloned into an expression vector pET30 (Novagen) to express Cas9 as described in earlier sections. Guide RNA sequences will be assembled into oligonucleotides and synthetized (IDT). Active combinations of Cas9 and guide RNA will be assessed by digestion of sequenced DNA templates at 37° C. (control) and at 37° C. after treatment at 95° C. for 3×5 min.
The second method is in vitro evolution of S. pyogenes Cas9. The sequence of Cas9 will be mutagenized to improve its thermostability. Briefly, this will be done by site-directed mutagenesis to remove excess loop sequences, increasing the number of ionic bridges between protein domains, or by diluting into droplets and PCR to create a pool of potential mutants. All mutants will be assessed for activity and thermostability as described above.
In the embodiments discussed, it is desirable to provide positive and/or negative controls. A positive control would ensure depletion of unwanted DNA is proceeding with fidelity and efficiency. A negative control would ensure that off-target cutting was minimal or nonexistent.
The positive control could consist of a sequencing library with inserts containing either all or most of the target sequences of the gRNAs included in the kit. This control would be run alongside a user's reaction to ensure that all components are working properly. After depletion with Cas9, elimination of the target sequences could be measured by gel electrophoresis or qPCR.
The negative control could consist of either 1) a set of gRNAs with less than 100% identity to the gRNAs used in the kit; for example, with 1, 2, 3, 4, 5, or more mismatches to the target-specific sequence; or 2) a DNA library having inserts with less than 100% identity to the targeted sequences of gRNAs used in the kit; for example, with 1, 2, 3, 4, 5, or more mismatches to the complementary sequence of the gRNA. This control would be run alongside a user's reaction to ensure that all components are working properly and to measure any off-target activity of the enzyme. After depletion with Cas9, the amount of off-target depletion could be measured by gel electrophoresis or qPCR.
Carpenter, Meredith L., Bustamante, Carlos D., Gourguechon, Stephane B.
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